Extracellular vesicles and methods of using

ABSTRACT

The present disclosure relates to materials and methods for extracellular vesicle (e.g., exosome)-mediated delivery of cargo (e.g., endogenous and/or exogenous) to non-bovine mammalian (e.g., human) cells. For example, exosomes isolated from bovine milk for delivering cargo to non-bovine mammalian (e.g., human) cells are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to United StatesProvisional Patent Application serial number U.S. Ser. No. 62/471,572,filed Mar. 15, 2017, the entirety of which is hereby incorporated byreference.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under DK107264 awardedby the National Institutes of Health; under 2015-67017-23181 and2016-67001-25301 awarded by the National Institute of Food andAgriculture; and under National Institute of General Medicine grant1P20GM104320. The government has certain rights in the invention.

TECHNICAL FIELD

This document relates to materials and methods for extracellularvesicle-mediated delivery of cargo to mammalian cells. For example, thisdocument provides exosomes isolated from milk for delivering cargo tomammalian cells.

BACKGROUND OF THE INVENTION

Exosomes, microvesicles, and apoptotic bodies are extracellular vesiclesdistinguished by size, biogenesis, and cargos. Exosomes contain diversecargos, and are involved in cell-to-cell communication.

SUMMARY

Encapsulation of cargos in exosomes protects the cargo against harshphysiological conditions such as low pH in the stomach, and againstexposure to enzymes such as RNases and proteases in the small intestineand during manufacturing, thereby conferring protection againstdegradation and providing a pathway for transport through thegastrointestinal tract.

The present disclosure relates to materials and methods forextracellular vesicle (e.g., exosome)-mediated delivery of cargo (e.g.,endogenous and/or exogenous) to mammalian (e.g., human) cells. In someembodiments, this disclosure provides exosomes isolated from milk, i.e.milk exosomes, for delivering cargo to non-bovine mammalian (e.g.,human) cells. In some embodiments, the exosomes are isolated from sheep,goat, camel, horse, donkey, reindeer, yak, buffalo, or bovine (cow) milkor colostrum.

As provided herein, exosome-rich preparations of extracellular vesiclesfrom milk are bioavailable in mammals, including humans. For example,milk exosomes administered to human intestinal cells and venousendothelial cells are taken up, and cargos present in such exosomes aresecreted or delivered into a receptor cell. The cellular uptake of themilk exosomes depends on surface glycoproteins on both the exosome andthe receptor cell. mRNAs (endogenous and exogenous) present in milkexosomes can be translated into peptides by cells to which they aredelivered. In some embodiments, the present disclosure provides a methodof altering the metabolism (e.g., the metabolism of purines and/or aminoacids) of a receptor cell; of increasing muscle strength; altering thegut microbiome (e.g., increasing or decreasing populations of particulargut flora); enhancing neurological processes (e.g., enhancing spatiallearning and memory, and/or sensorimotor gating); or increasingfertility, comprising the step of administering an effective amount ofmilk exosomes loaded with a cargo to a mammal in need thereof. In someembodiments, the exosomes are isolated from bovine milk or colostrum.

Having the ability to deliver exosomal cargo to recipient cells providesa unique and unrealized opportunity to deliver exosomal (e.g.,endogenous and/or exogenous) cargo.

In some embodiments, the disclosure provides a milk exosome comprising abiological membrane surrounding a lumen, wherein the biological membranecomprises one or more glycoprotein(s), wherein the biological membraneis modified as compared with the natural biological membrane of the milkexosome. In some embodiments, the biological membrane is modified suchthat it has an increased number of one or more of its nativeglycoprotein(s). In some embodiments, the biological membrane ismodified such that it has a decreased number of one or more of itsnative glycoprotein(s). In some embodiments, the exosome is producedusing an enzyme selected from a serine protease, cysteine protease ormetalloprotease. In some embodiments, the enzyme is selected fromtrypsin, AspN, GluC, ArgC, chymotrypsin, proteinase K, and Lys-C. Insome embodiments, the biological membrane is modified such that one ormore of its native glycoprotein(s) is not present. In some embodiments,the biological membrane is modified such that it includes one or moreglycoprotein(s) that is not naturally present in the natural biologicalmembrane.

In some embodiments of the present disclosure, the biological membraneof the exosome is modified such that one or more of its nativeglycoprotein(s) is altered. In some embodiments, the one or more nativeglycoprotein(s) is altered such that the number of glycan residuespresent on the glycoprotein(s) is increased. In some embodiments, theexosome is produced using glycosylation that adds one or more glycans tothe glycoprotein. In some embodiments, the one or more nativeglycoprotein(s) is altered such that the number of glycan residuespresent on the glycoprotein(s) is decreased. In some embodiments, thenumber of glycan residues is decreased by cleavage of one or more glycanresidues present on the glycoprotein(s). In some embodiments, theexosome is produced using an enzyme selected from a glycosidase,exoglycosidase, endoglycosidase, glycoamidase, neuraminidase,galactosidase, peptide:N-glycosidase (PNGase), glycohydrolase, and anycombination thereof. In some embodiments, the enzyme is selected from aβ-N-acetylglucosaminidase, PNGase F, β (1-4) Galactosidase,O-Glycosidase, N-Glycosidase, N-glycohydrolase, Endo H, Endo D, Endo F₂,EndoF₃, and any combination thereof. In some embodiments, two or morenative glycoprotein(s) are altered such that at least one glycoproteinhas an increased number of glycan residues and at least one otherglycoprotein has a decreased number of glycan residues or is missing itsglycan residue(s), wherein the glycoprotein(s) having an increasednumber of glycan residues is different from the glycoprotein(s) having adecreased number of glycan residues or missing glycan residues. In someembodiments, the one or more native glycoprotein(s) is altered such thatit comprises a modified glycan. In some embodiments, the modified glycancomprises at least one carbohydrate moiety that differs from that of theglycan in the native glycoprotein(s). In some embodiments, the modifiedglycan comprises one or more galactose, mannose, O-glycans,N-acetyl-glucosamines, and/or N-glycan chains or any combinationthereof. In some embodiments, the glycan is selected from comprises oneor more D- or L-glucose, erythrose, fucose, galactose, mannose, lyxose,gulose, xylose, arabinose, ribose, 2′-deoxyribose, glucosamine,lactosamine, polylactosamine, glucuronic acid, sialic acid, sialyl-LewisX (SLex), N-acetyl-glucosamine, N-acetyl-galactosamine, neuraminic acid,N-glycolylneuraminic acid (Neu5Gc), N-acetylneuraminic acid (Neu5Ac), anN-glycan chain, an O-glycan chain, a Core 1, Core 2, Core 3, or Core 4structure, or a phosphate- or acetate-modified analog thereof or acombination thereof. In some embodiments, the modified glycan lacks aportion of one or more of its carbohydrate chain(s). In someembodiments, the modified glycan is missing one or more of itscarbohydrate chain(s). In some embodiments, the modified glycancomprises one or more altered carbohydrate chain(s). In someembodiments, the one or more native glycoprotein(s) is altered such thatat least one glycan present on the glycoprotein(s) is substituted with aglycan that is not naturally present in the native glycoprotein(s). Insome embodiments, the one or more native glycoprotein(s) is altered byblocking one or more glycan residue(s) present on the glycoprotein(s).In some embodiments, the one or more glycan residue(s) is blocked bylectin binding to the glycan residue. In some embodiments, the lectin isselected from Concanavalin A, Lentil lectin, Snowdrop lectin, Ricin(Ricinus communis Agglutinin, RCA120), Peanut agglutinin, Jacalin, Hairyvetch lectin, Dolichos biflorus agglutinin, Soybean agglutinin,N-acetylglucosamine binding lectins, Wheat Germ Agglutinin (WGA),Phaseolus vulgaris agglutinin, Elderberry lectin, Maackia amurensisleukoagglutinin, Maackia amurensis hemoagglutinin, Ulex europaeusagglutinin, or Aleuria aurantia lectin.

In some embodiments of the present disclosure, the uptake of the milkexosome into a mammalian cell is altered as compared with the uptake ofa corresponding milk exosome having its natural biological membrane. Insome embodiments, the uptake of the milk exosome into a mammalian cellis increased. In some embodiments, the uptake of the milk exosome into amammalian cell is decreased. In some embodiments, the mammalian cell isselected from an intestinal cell, venous endothelial cell or otherendothelial cell, immune cell, macrophage, intestinal mucosa, peripheralcell of the liver, spleen, lung, brain, kidneys, or pancreas, cancercell, or fetal cell. In some embodiments, the cell is a human cell.

In some embodiments of the present disclosure, the milk exosome istargeted to a specific mammalian cell or tissue. In some embodiments,the mammalian cell is selected from an intestinal cell, venousendothelial cell or other endothelial cell, immune cell, macrophage,intestinal mucosa, peripheral cell of the liver, spleen, lung, brain,kidneys, or pancreas, cancer cell, or fetal cell. In some embodiments,the mammalian tissue is selected from liver, spleen, lung, brain,kidneys, pancreas, gastrointestinal tract, small intestine, colon,stomach, heart.

In some embodiments of the present disclosure, degradation of theexosome by macrophages is reduced as compared with an exosome having itsnatural biological membrane. In some embodiments, the stability of theexosome in the gastrointestinal tract, systemic circulation, lymphaticcirculation, intracellular conditions, or other tissues or organs of ahuman is increased as compared with an exosome having its naturalbiological membrane. In some embodiments, the stability of the exosomeunder physiological conditions in a human is increased as compared withan exosome having its natural biological membrane. In some embodimentsof the present disclosure, the exosome further comprises an exogenouscargo encapsulated in said lumen. In some embodiments, the exosomefurther comprises a miRNA or mRNA that is biologically active in amammal. In some embodiments, the exosome is isolated from sheep, goat,camel, horse, donkey, reindeer, yak, buffalo, or bovine (cow) milk orcolostrum. In some embodiments, the exogenous cargo is selected from oneor more nucleic acid molecules, polypeptides, lipids, vitamins,minerals, small molecules, pharmaceuticals, hormones, or enzymes. Insome embodiments, the exogenous cargo comprises a therapeutic agent. Insome embodiments, the therapeutic agent is selected from mRNAs,polypeptides, miRNAs, miRNA antagonists, nutrients, antibiotics, cancerdrugs, activators of Toll-like receptors, or molecules capable ofdelivery to macrophages. In some embodiments, the therapeutic agent is acancer drug selected from a chemotherapeutic, an immunotherapeutic, ahormone therapeutic, or a targeted therapeutic. In some embodiments, theexogenous cargo comprises a nutritional agent. In some embodiments, thenutritional agent is selected from vitamins, minerals, lipids, fattyacids, mRNAs, or polypeptides. In some embodiments, the nutritionalagent is a fatty acid selected from omega-3 fatty acids or omega-6 fattyacids.

In some embodiments the disclosure provides a composition formulated fororal administration to a human, said composition comprising any of theexosome described herein. In some embodiments, the exosome comprises anendogenous cargo. In some embodiments, the exosome comprises anexogenous cargo.

In some embodiments the present disclosure provides an infant formula ornutritional supplement, said infant formula or nutritional supplementcomprising any of the exosomes described herein. In some embodiments,the exosome in the infant formula or nutritional supplement comprises anendogenous cargo. In some embodiments, the exosome in the infant formulaor nutritional supplement comprises an exogenous cargo. In someembodiments, the exosome in the infant formula or nutritional supplementcomprises one or more nutritional agents selected from vitamins,minerals, lipids, fatty acids, mRNAs, or polypeptides.

In some embodiments the present disclosure provides a method of alteringthe uptake of a milk exosome into a mammalian cell or tissue, saidexosome having a biological membrane comprising one or moreglycoprotein(s), comprising modifying the biological membrane of theexosome. In some embodiments, the uptake of the milk exosome into amammalian cell or tissue is increased. In some embodiments, the uptakeof the milk exosome into a mammalian cell or tissue is decreased. Insome embodiments, the uptake of the milk exosome into a mammalian cellor tissue is selectively increased in a targeted mammalian cell ortissue. In some embodiments, the uptake of the milk exosome into amammalian cell or tissue is selectively decreased in a targetedmammalian cell or tissue. In some embodiments the present disclosureprovides a method of targeting a milk exosome to a selected mammaliancell or tissue, said exosome having a biological membrane comprising oneor more glycoprotein(s), comprising modifying the biological membrane ofthe exosome.

In some embodiments of these methods, the biological membrane ismodified such that it has an increased number of one or more of itsnative glycoprotein(s). In some embodiments of these methods, thebiological membrane is modified such that it has a decreased number ofone or more of its native glycoprotein(s). In some embodiments of thesemethods, the exosome is produced using an enzyme selected from a serineprotease, cysteine protease or metalloprotease. In some embodiments ofthese methods, the enzyme is selected from trypsin, AspN, GluC, ArgC,chymotrypsin, proteinase K, and Lys-C. In some embodiments of thesemethods, the biological membrane is modified such that one or more ofits native glycoprotein(s) is not present. In some embodiments of thesemethods, the biological membrane is modified such that it includes oneor more glycoprotein(s) that is not naturally present in the naturalbiological membrane. In some embodiments of these methods, thebiological membrane is modified such that one or more of its nativeglycoprotein(s) is altered. In some embodiments of these methods, theone or more native glycoprotein(s) is altered such that the number ofglycan residues present on the glycoprotein(s) is increased. In someembodiments of these methods, the one or more native glycoprotein(s) isaltered such that the number of glycan residues present on theglycoprotein(s) is decreased. In some embodiments of these methods, thenumber of glycan residues is decreased by cleavage of one or more glycanresidues present on the glycoprotein(s) using an enzyme selected from aglycosidase, exoglycosidase, endoglycosidase, glycoamidase,neuraminidase, galactosidase, peptide:N-glycosidase (PNGase),glycohydrolase, and any combination thereof. In some embodiments ofthese methods, the enzyme is selected from a β-N-acetylglucosaminidase,PNGase F, β (1-4) Galactosidase, O-Glycosidase, N-Glycosidase,N-glycohydrolase, Endo H, Endo D, Endo F₂, EndoF₃, and any combinationthereof. In some embodiments of these methods, two or more nativeglycoprotein(s) are altered such that at least one glycoprotein has anincreased number of glycan residues and at least one other glycoproteinhas a decreased number of glycan residues or is missing its glycanresidue(s), wherein the glycoprotein(s) having an increased number ofglycan residues is different from the glycoprotein(s) having a decreasednumber of glycan residues or missing glycan residues. In someembodiments of these methods, the one or more native glycoprotein(s) isaltered such that it comprises a modified glycan. In some embodiments ofthese methods, the modified glycan comprises one or more D- orL-glucose, erythrose, fucose, galactose, mannose, lyxose, gulose,xylose, arabinose, ribose, 2′-deoxyribose, glucosamine, lactosamine,polylactosamine, glucuronic acid, sialic acid, sialyl-Lewis X (SLex),N-acetyl-glucosamine, N-acetyl-galactosamine, neuraminic acid,N-glycolylneuraminic acid (Neu5Gc), N-acetylneuraminic acid (Neu5Ac), anN-glycan chain, an O-glycan chain, a Core 1, Core 2, Core 3, or Core 4structure, or a phosphate- or acetate-modified analog thereof or acombination thereof and wherein the modified glycan lacks a portion ofone or more of its carbohydrate chain(s), is missing one or more of itscarbohydrate chain(s), or comprises one or more altered carbohydratechain(s). In some embodiments of these methods, the one or more nativeglycoprotein(s) is altered such that at least one glycan present on theglycoprotein(s) is substituted with a glycan that is not naturallypresent in the native glycoprotein(s). In some embodiments of thesemethods, the one or more native glycoprotein(s) is altered by blockingone or more glycan residue(s) present on the glycoprotein(s). In someembodiments of these methods, the one or more glycan residue(s) isblocked by lectin binding to the glycan residue. In some embodiments ofthese methods, the lectin is selected from Concanavalin A, Lentillectin, Snowdrop lectin, Ricin (Ricinus communis Agglutinin, RCA120),Peanut agglutinin, Jacalin, Hairy vetch lectin, Dolichos biflorusagglutinin, Soybean agglutinin, N-acetylglucosamine binding lectins,Wheat Germ Agglutinin (WGA), Phaseolus vulgaris agglutinin, Elderberrylectin, Maackia amurensis leukoagglutinin, Maackia amurensishemoagglutinin, Ulex europaeus agglutinin, or Aleuria aurantia lectin.In some embodiments of these methods, the mammalian cell is selectedfrom an intestinal cell, venous endothelial cell or other endothelialcell, immune cell, macrophage, intestinal mucosa, peripheral cell of theliver, spleen, lung, brain, kidneys, or pancreas, cancer cell, or fetalcell. In some embodiments of these methods, the cell is a human cell. Insome embodiments of these methods, the mammalian tissue is selected fromliver, spleen, lung, brain, kidneys, pancreas, gastrointestinal tract,small intestine, colon, stomach, or heart.

In some embodiment, the present disclosure provides a method ofcorrecting dysbiosis or improving the gut microbiome or gut health of asubject, comprising administering to said subject an effective amount ofany of the exosomes described herein. In some embodiments, thecorrecting dysbiosis or improving the gut microbiome or gut health of asubject comprises a decrease in Ruminococcaceae and/or Verrucomicrobiae.In some embodiments, the correcting dysbiosis or improving the gutmicrobiome or gut health of a subject comprises an increase inClostridiales or Erysipelotrichaceae.

In some embodiments, the present disclosure provides a method oftreating inflammatory bowel disease in a subject, comprisingadministering to a subject in need thereof an effective amount of any ofthe exosome provided herein. In some embodiments, the treatinginflammatory bowel disease in a subject comprises an increase inLachnospiraceae and Ruminococcaceae. In some embodiments, the treatinginflammatory bowel disease in a subject comprises a decrease inEnterobacteriaceae.

In some embodiments, the present disclosure provides a method oftreating obesity in a subject, comprising administering to a subject inneed thereof an effective amount of any of the exosomes describedherein. In some embodiments, the treating obesity in a subject comprisesa decrease in the ratio of Firmicutes and Bacteroidetes. In someembodiments, the treating obesity in a subject comprises a decrease inthe ratio of Firmicutes and Bacteroidetes.

In some embodiments, the present disclosure provides a method oftreating non-alcoholic fatty liver in a subject, comprisingadministering to a subject in need thereof an effective amount of any ofthe exosomes described herein. In some embodiments, the treatingnon-alcoholic fatty liver in a subject comprises an increase inRuminococcaceae and Escherichia.

In some embodiments, the present disclosure provides a method ofincreasing muscle strength, enhancing sensorimotor gating or cognitiveperformance, or increasing fertility or fecundity in a subject,comprising administering to said subject an effective amount of any ofthe exosomes described herein or any of the nutritional supplementsdescribed herein. In some embodiments, the present disclosure provides amethod of treating sarcopenia, muscle loss after injury,atherosclerosis, cancer, an immune disease, impaired fecundity, orcognitive impairment, comprising administering to a subject in needthereof any of the exosomes provided herein or any of the nutritionalsupplements described herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Methods and materials aredescribed herein for use in the present disclosure; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings, description, and the claims. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 is a schematic depicting an exosome that includes variousexemplary cargos, surface proteins, and other associated biologicalmolecules.

FIGS. 2A and 2B show milk exosome preparations from cow's milk. FIG. 2Ashows exosome extracts probed using anti-CD63, anti-CD9, anti-Alix,anti-α-s1 casein, and antihistone H3. Protein extracts were run on thesame gel, membranes were cut for probing with the three antibodies, andimages were reassembled after probing. FIG. 2B shows transmissionelectron microscope images of exosome preparations. The large fieldimage was obtained with a 15,000-fold magnification; the insert depictsa single particle selected from the same image. M, molecular weightmarkers; E, exosome extract.

FIGS. 3A-D show exosome cytoplasmic extract vs. exosome membrane proteincharacterization of membrane proteins isolated from milk exosomes. FIG.3A shows GADPH expression in the cytoplasmic extract. FIG. 3B shows ALIXexpression in the exosome membrane protein extract (ALIX is a marker forexosomes). FIG. 3C shows CD63 expression in the membrane protein extract(CD63 is a marker for exosomes). FIG. 3D shows CD9 expression in theexosome membrane protein extract (CD9 is a marker for exosomes).

FIG. 4 shows characterization assay results for bovine milk exosomes.Rabbit anti-bovine α-s1 casein and gel electrophoresis were used toprobe membrane blots of 1) cow's milk exosomes, 2) cow's milk, 3) humanbreast milk, 4) platelet glycoprotein 1 synthetic peptide, 5) α-s1casein peptide, and 6) chicken egg yolk exosomes. Ten micrograms of milkand exosome protein were loaded per lane, whereas only 1 μg of syntheticpeptides were loaded. M, molecular weight markers.

FIG. 5 shows nano tracking analyzer size analysis of milk exosomes.

FIG. 6 shows nano tracking analyzer size analysis of sonicated milkexosomes.

FIGS. 7A and 7B show control/normal (non-sonicated) bovine milk exosomeatomic force microscopy results. FIG. 7A shows that exosomes haveindividual, smooth and well-formed surfaces. FIG. 7B is a graphicalrepresentation of the range of diameters for non-sonicated exosomes.

FIGS. 8A and 8B show atomic force microscopy results for sonicatedbovine milk exosomes. FIG. 8A shows that in comparison to the normalexosomes, sonicated exosomes have rough edges and some clustering oraggregation (black arrow points on the 3D images of exosomes). FIG. 8Bis a graphical representation of the range of diameters for sonicatedexosomes.

FIGS. 9A and 9B show the effect of sonication treatment on the uptake ofexosomes in cells. FIG. 9A shows the uptake of non-sonicated andsonicated exosomes into Caco-2 cells. FIG. 9B shows the uptake ofnon-sonicated and sonicated exosomes into FH cells (n=3; *Different fromcontrol, P<0.05. Values are means±S.D).

FIG. 10 shows the effect of sonication treatment on the uptake ofexosomes in U937 cells (n=3). *Different from control, P<0.05. Valuesare means±S.D.

FIGS. 11A and 11B show the uptake of milk exosomes loaded with puromycin(Puro) or enhanced green fluorescent protein (eGFP) plasmid,respectively, into HUVEC cells. FIG. 11A shows the effects ofpuromycin-loaded milk exosomes on HUVEC survival. Cells were treatedwith puromycin-free exosomes (left bar), puromycin-loaded exosomes(middle bar), and free puromycin (right bar) (^(a,b) P<0.05 for bars notsharing a common letter). FIG. 11B shows the expression of eGFP after 3days of HUVEC culture with eGFP plasmid-loaded exosomes.

FIGS. 12A and B show uptake of milk exosomes loaded with puromycin(Puro) or enhanced green fluorescent protein (eGFP) plasmid,respectively. FIG. 12A shows the effects of puromycin-loaded milkexosomes on HUVEC survival. Cells were treated with puromycin-freeexosomes (left bar), puromycin-loaded exosomes (middle bar), and freepuromycin (right bar) (^(a,b) P<0.05 for bars not sharing a commonletter). FIG. 12B shows expression of eGFP after 3 days of HUVEC culturewith eGFP plasmid-loaded exosomes.

FIGS. 13A and B show super resolution microscopy results. The nuclei ofthe cell stained with Hoechst-blue, cytosol (f-Actin) with CF-594-redand milk exosomes with PKH-67-green. FIG. 13A shows a split image foronly cells, only exosomes (top left and right panels, respectively),only nucleus and both exosomes and cells (bottom left and right panels,respectively). FIG. 13B is a orthogonal cross sectional image showingthe presence of exosome in the cytosol after its uptake by the cells.

FIGS. 14A-C show DiR-labeled exosomes (FIG. 14A) and exo-GLOW redlabeled RNA in exosomes (FIG. 14B) in excised tissues 12 hours afteroral gavage. Also shown (FIG. 14C) are Exo-GLOW Red labeled RNA inexosomes in excised tissues 24 hours after oral gavage.

FIG. 15 is a graphical representation showing the signal density ofExo-GLOW Red labeled RNA in exosomes in excised tissues (liver, spleen,kidneys, heart, lungs, and brain) 12 hours after oral gavage.

FIGS. 16A and B show DiR-labeled exosomes in excised tissues 24 hoursafter oral gavage (FIG. 16A) and Exo-GLOW Red labeled RNA in exosomes(FIG. 16B) in excised tissues 24 hours after gavage.

FIG. 17 shows the effects of exosome-defined diets on fertility in mice.Exosome-depleted (Exo−) diet mice had lower fertility thanexosome-sufficient (Exo+) mice, particularly when both males and femaleswere fed the Exo+ diet.

FIG. 18 shows the effects of Exo- and Exo+ diets on litter size in mice.

FIG. 19 shows the effects of Exo- and Exo+ diets on the survival of pupswhose parents were fed exosome-defined diets.

FIGS. 20A-E show that hepatic concentrations of amino acids were up to1800% higher in mice fed an exosome-depleted (Exo−) diet compared withmice fed an exosome-sufficient (Exo+) diet (control) for 4 weeks. N=8,*p<0.05 vs. Exo+. FIG. 20A shows the abundance of leucine in mice fedthe Exo− versus Exo+ diet. FIG. 20B shows the abundance of phenylalaninein mice fed the Exo− versus Exo+ diet. FIG. 20C shows the abundance ofalanine in mice fed the Exo− versus Exo+ diet. FIG. 20D shows theabundance of leucine-lysine dipeptide metabolite in mice fed the Exo−versus Exo+ diet. FIG. 20E shows the abundance of glutamyl-isoleucinedipeptide metabolite in mice fed the Exo− versus Exo+ diet.

FIGS. 21A and 21B show that the mRNA expression of branched chain aminoacid (BCAA) transporter 1 (cytoplasm, BCAT1, FIG. 21A) and BCAAtransporter 2 (mitochondria, BCAT2, FIG. 21B) was greater in mice fed anExo− diet compared to mice fed an Exo+(n.s. for BCAT2). Expression ofBCAA transporter mRNAs is shown in livers of C57BL/6 mice fed an Exo− orExo+(control) diet for 4 weeks. N=8, *p<0.05 vs. Exo+.

FIG. 22 shows latency to enter the escape hole in a Barnes maze byC57BL/6 mice fed bovine milk exosome-defined diet. Shown are Mean±SEM.N=5 per group. *p<0.05 vs. E+. Abbreviations: F, female; M, male; E+,Exo+ fed mice; Exo−, Exo− fed mice.

FIG. 23 shows latency to locate the escape platform in a Morris watermaze by C57BL/6 mice fed bovine milk exosome-defined diet. Shown areMean±SEM. N=5 per group. *p<0.05 vs. E+. Abbreviations: F, female; M,male, E; Exo+ fed mice; Exo−, Exo− fed mice.

FIG. 24 shows effects of bovine milk exosome-defined diet on Prepulseinhibition (PPI) in C57BL/6 mice (68 dB prepulse intensity, 105 dBstartle intensity). Shown are Mean±SEM. N=5 per group. *p<0.05 vs. E+.Abbreviations: F, female; M, male; E; Exo+ fed mice; Exo−, Exo− fedmice.

FIG. 25 shows different abundance of Operational Taxonomic Units (OTUs)in mice fed exosome-defined diets at age 15 weeks. For example, therelative abundance of Firmicute classes Clostridia (Ruminococcaceae) andVerrucomicrobia classes Verrucomicrobiae (Muciniphila) were greater inmice fed Exo− compared with Exo+at age 15 weeks, whereas the relativeabundance of Firmicute classes Clostridia (Clostridiales) was smaller inmice fed Exo-compared with Exo+at age 45 weeks (see FIG. 26).

FIG. 26 shows different abundance of OTUs in mice fed exosome-defineddiets at age 45 weeks.

FIG. 27 shows alpha diversity (Chao1) and Beta diversity (PrincipalCoordinates Analysis) in the cecum of mice fed exosome RNA-sufficient(ERS) or exosome RNA-depleted (ERD) diets at ages 7, 15 and 47 weeks.

FIG. 28 shows microbial phyla in the cecum of mice fed exosomeRNA-sufficient (ERS) or exosome RNA-depleted (ERD) diets at ages 7, 15and 47 weeks. Values represent average percent relative abundance acrossall samples. F, female; M, male.

FIG. 29 shows microbial families in the cecum of mice fed exosomeRNA-sufficient (ERS) or exosome RNA-depleted (ERD) diets at ages 7, 15and 47 weeks. o, order; f, family; p, phylum.

FIG. 30 shows microbial operational taxonomic units (OTUs) in the cecumof mice fed exosome RNA-sufficient (ERS) or exosome RNA-depleted (ERD)diets at ages 7, 15 and 47 weeks. Effects of diet were statisticallysignificant (P<0.05), if the log score in the linear discriminantanalysis (LDA) was greater than 2.

FIG. 31 shows the correlation between changes in OTUs with changes inthe hepatic transcriptome in female mice, age 15 weeks fed exosomeRNA-sufficient (ERS) or exosome RNA-depleted (ERD).

FIG. 32 shows KEGG pathway analysis for metabolic functions that areenriched in the hepatic transcriptome in female mice, age 15 weeks fedexosome RNA-sufficient (ERS) or exosome RNA-depleted (ERD).

FIGS. 33 and 33B show time courses of bovine exosome uptake in Caco-2cells and IEC-6 cells. FIG. 33A shows exosome uptake into human coloncarcinoma Caco-2 cells as a function of time at a concentration of 110mg exosome protein/200 mL media and a temperature of 37° C. (n=6). FIG.33B shows exosome uptake into rat primary intestinal IEC-6 cells as afunction of time at a concentration of 55 mg exosome protein/200 mLmedia and a temperature of 37° C. (n=3). Values are means 6 SDs.

FIGS. 34A-34C show temporal kinetics of milk exosome uptake in mammaliancells. FIG. 34A shows the uptake of exosomes into human umbilical veinendothelial cells (HUVECs) over 480 minutes (4 hours) using 20 μgexosome protein/200 μl of media. The insert in FIG. 34A illustrates thetemporal pattern for 120 min (2 hours): y=0.0003×+0.009, R²=0.98. FIG.34B shows a milk exosome uptake study in Caco-2 cells over the course of8 hours in which N=3; p<0.05. FIG. 34C shows an exosome uptake study inhuman small intestinal cells (FHs cells) over the course of 8 hours.

FIGS. 35A and 35B show saturation kinetics of bovine exosome transportin intestinal cells. FIG. 5A shows exosome uptake into human coloncarcinoma Caco-2 cells as a function of substrate concentration at 37°C. (n=5). FIG. 5B shows exosome uptake into rat primary small intestinalIEC-6 cells as a function substrate concentration at 37° C. (n=3).Values are means 6 SDs.

FIGS. 36A and 36B show saturation kinetics of milk exosome uptake inCaCo2 and HUVEC cells, respectively. FIG. 36A shows exosome uptake intohuman colon carcinoma Caco-2 cells as a function of substrateconcentration at 37° C. (N=3; p<0.05). FIG. 36B shows exosome uptakeinto human umbilical vein endothelial cells as a function of substrateconcentration at 37° C.

FIGS. 37A and 37B show saturation kinetics of milk exosome uptake inhuman small intestinal cells (FHs cells). FIG. 37A shows exosome uptakeinto FH cells as a function of substrate concentration at 37° C.(Y=0.008327*X−0.06516; R²=0.9747). FIG. 37B shows another graphicalrepresentation of the experiment shown in FIG. 37A.

FIG. 38 shows a scheme for exosome processing to remove glycoproteins onthe surface of milk exosomes and also to remove glycans from the surfaceglycoproteins. These methods were used to generate the data shown inFIGS. 4-43, 46-49, 51, 52.

FIG. 39 is a graphical representation showing the cleavage sites ofvarious proteases. Trypsin cleaves at lysine or arginine residues;chymotrypsin cleaves at aromatic amino acids (phenylalanine, tryptophan,and tyrosine); lys-C cleaves at lysine; Arg-C cleaves at arginine andlysine residues; Glu-C cleaves at glutamic acid and aspartic acidresidues; and Asp-N cleaves at aspartic acid residues.

FIGS. 40A-C show the effect on exosome uptake by different cells afterremoval of surface proteins from the exosomes or the cells. FIG. 40Ashows the effect on exosome uptake after treatment of milk exosomes orHUVEC cells with proteinase K (FIG. 40A). FIG. 40B shows the effect onexosome uptake after treatment of milk exosomes with trypsin or Glu-C(FIG. 40B) or Arg-C or Asp-N FIG. 40C). N=3, *P<0.05.

FIGS. 41A and 41B show exosome uptake by Caco-2 cells after treatmentswith the indicated protease(s) (n=3; p<0.05). Treatment of exosomes(FIG. 41A) with protease decreases the uptake of cow's milk exosomes incells. Treatment of Caco-2 cells (FIG. 41B) with protease decreases theuptake of cow's milk exosomes in cells. *P<0.05 vs. control. (N=3,means±S.D.).

FIGS. 42A and 42B show an exosome uptake study in human small intestinalcells (FHs cells) after treatment with the indicated proteases (n=3;p<0.05). Treatment of exosomes (FIG. 42A) with proteases decreases theuptake of cow's milk exosomes in FH cells. Treatment of FH cells withproteases (FIG. 42B) decreases the uptake of cow's milk exosomes in FHcells. *P<0.05 vs. control. (N=3, means±S.D.).

FIGS. 43A and 43B show an exosome uptake study in human macrophage U937cells after treatment with the indicated proteases (n=3; p<0.05).Treatment of exosomes (FIG. 43A) with proteases decreases the uptake ofcow's milk exosomes in U937 cells. Treatment of U937 cells withproteases (FIG. 43B) decreases the uptake of cow's milk exosomes in U937cells. *P<0.05 vs. control. (N=3, means±S.D.).

FIG. 44 shows a table of exemplary enzyme treatments, expected number ofTMHs, and predicted number of binding sites on exosome surface proteins.T.D.=Total Deglycosylation. TMHs=Transmembrane Helix.

FIG. 45 shows a Venn diagram comparison for identified membrane proteinsafter specific protease treatment versus total glycan removal andspecific protease treatment.

FIG. 46 shows exosome uptake in Caco2 cells following enzymatic removalof glycan from exosome cell surface proteins using PNGase,β-galactosidase, O-glycosidase, N-acetyl-glucosamidase, or a mixturethereof. Removal of glycan results in a decrease in exosome uptake inCaco-2 cells. LC/MS-MS: 4 N-, 2 O-, and 2 C-glycosylated proteins wereidentified on the milk exosome surface.

FIGS. 47A and 47B show exosome uptake by Caco-2 cells after treatmentswith various glycosidase(s) (β-N Acetylglucosaminidase, PngaseF, β(1→4Galactosidase, O-Glycosidase, Neuraminidase, and mixed glycosidases)(n=3; p<0.05). Treatment of exosomes (FIG. 47A) with glycosidasedecreases the uptake of cow's milk exosomes in Caco-2 cells. Treatmentof Caco-2 cells (FIG. 47B) with glycosidase decreases the uptake ofcow's milk exosomes in cells. *P<0.05 vs. control. (N=3, means±S.D.).FIG. 48 shows exosome uptake by human small intestinal cells (FHs cells)after treatments with various glycosidases (β-N Acetylglucosaminidase,PngaseF, β(1→4 Galactosidase, O-Glycosidase, Neuraminidase, and mixedglycosidases). (n=3; p<0.05). Treatment of exosomes with glycosidasedecreases the uptake of cow's milk exosomes in FH cells. *P<0.05 vs.control. (N=3, means±S.D.).

FIGS. 49A and 49B show exosome uptake by U937 cells after treatmentswith various glycosidases (β-N Acetylglucosaminidase, PngaseF, β(1→4Galactosidase, O-Glycosidase, Neuraminidase, and mixed glycosidases).Treatment of exosomes (FIG. 49A) with glycosidase decreases the uptakeof cow's milk exosomes in U937 cells. Treatment of U937 cells (FIG. 49B)with glycosidase decreases the uptake of cow's milk exosomes in the U937cells. *P<0.05 vs. control. (N=3, means±S.D.).

FIGS. 50A-D shows Eastern/Lectin Blots that identify glycans present onmembranes of milk exosomes. NE—Normal Exosome; CE—Cytoplasmic Extract;MP—Membrane Protein; FIG. 50A shows a blot using lectin Con A(Concanavalin A) as a probe which is specific for alpha linked mannose;FIG. 50B shows a blot using lectin PNA (Peanut agglutinina) as a probe,which is specific for Gal β 1-3 GaNAc 1 Ser/Thr; FIG. 50C shows a blotusing lectin SBA (Soybean agglutinin) as a probe which is specific forGaNAc; FIG. 50D shows a blot using lectin SNA (Elderberry lectin) as aprobe which is specific for sialic acid. These results demonstrate thepresence of alpha linked mannose, Gal β 1-3 GalNAc 1 Ser/Thr, GalNAc,and sialic acid glycosylation on exosome membrane proteins.

FIGS. 51A and 51B show results of a lectin blocking study. Blocking ofglycans present on exosomes (FIG. 51A) and FHs cells (FIG. 51 B) withlectin decreases the uptake of cow's milk exosomes in cells. (*P<0.05vs. control; N=3, means±S.D.)

FIG. 52 shows the results of an exosome transport study in whichinhibitors of endocytosis (cytochalasin D=Cyt D), vesicle trafficking(brefeldin A=BFA), and carbohydrate blockage (glucose, galactose) wereshown to cause a decrease in exosome uptake in HUVEC cells.

FIGS. 53A and 53B show the design of a transgenic mouse that is atamoxifen-inducible conditional glycan transferase knockout mouse(homozygous). FIG. 53A shows that floxed B6 mice were crossed withB6;129-Bmi1^(tm1(cre/ERT)Mrc)/J mice to generate a homozygoustamoxifen-inducible conditional glycan transferase knockout mouse. FIG.53B shows the Cre-LoxP mediated gene deletion method used to knock-outthe glycan transferase gene.

FIGS. 54A and 54B show various gene knockouts which result in alterationof glycosylation and corresponding genotyping results. FIG. 54A providesa table showing glycosylation enzyme and corresponding glycan loss invarious knockout mouse strains. FIG. 54B shows genotyping resultsshowing knockout of the MGAT gene and knockout of the PoFUT1 gene.

FIGS. 55A and 55B depict the experimental process for administeringfluorescence-labelled exosomes to mice and the correspondingfluorescence results between unmodified and PNGase F-treated milkexosomes. FIG. 55A shows a scheme for exosome processing to removecertain glycans from membrane proteins on the surface of exosomes, e.g.,asparagine-linked complex, hybrid, or high mannose oligosaccharides (viathe use of PnGase F). These methods were used to generate the data shownin FIG. 55B. FIG. 55B shows the results of a fluorescence study in whichfluorescent-labelled exosomes having native glycosylation (control)versus altered glycosylation (removal of asparagine-linked complex,hybrid, or high mannose oligosaccharides via the use of PnGase F) werecompared.

FIG. 56 shows approaches for elucidating the biological roles ofglycans. The figure assumes that a specific biological role is beingmediated by recognition of a certain glycan structure by a specificglycan-binding protein. Clues to this biological role could be obtainedby a variety of different approaches.

FIGS. 57A and 57B show images demonstrating that sonication of exosomescaused a loss of bioavailability to non-detectable levels after oraladministration (FIG. 57A). Removal of exosomal surface proteins bytreatment with trypsin caused a reduced accumulation of exosomes in theliver and lungs after intravenous injection (FIG. 57B). The images showdistribution of sonicated and trypsinized exosomes in Balb/c mice. FIG.57A shows fluorescence signal in excised tissues from Balb/c mice 24hours after oral gavage of sonicated and DiR-labeled, unsonicated andDiR-labeled, or unlabeled and sonicated (1×1012/g body weight, n=3).FIG. 57B shows distribution of trypsinized DiR-labeled exosomes(1×1012/g body weight) in Balb/c mice in excised tissues 24 hours afteroral gavage (n=2).

FIG. 58 shows distribution of exosomes in macrophage-depleted mice.Balb/c mice were treated with clodronate (150 ul) by intraperitonealinjection to ablate endogenous macrophage populations. Unlabeled orDiR-labeled exosomes were administered by oral gavage 24 hours afterclodronate treatment, and tissues were harvested 24 hours after exosomeadministration for fluorescence analysis.

DETAILED DESCRIPTION

The present disclosure relates to materials and methods forextracellular vesicle-mediated delivery of cargo to mammalian cells. Insome embodiments, the present disclosure provides milk exosomes fordelivering cargo to a mammal (e.g., human). This disclosure alsoprovides methods of using an extracellular vesicle (e.g., exosome), suchas any of the vesicles described herein, to deliver one or more exosomalcargos to a recipient cell. For example, an exosome can be administeredto a mammal (e.g., a human) to alter the gut microbiome of a mammal, toregulate (e.g., increase or decrease) the immune response of a mammal,to enhance the fertility of a mammal, to alter (e.g., increase ordecrease) the metabolism of a mammalian cell, to alter (e.g., increaseor decrease) the gene expression of a mammalian cell, to increase themuscle strength of a mammalian cell, to enhance neurological processesof a mammal, and/or to treat a mammal having a disease. In someembodiments, an exosome can be loaded with an exogenous cargo (e.g., atherapeutic agent) and used to deliver the exogenous cargo to amammalian receptor cell.

In one aspect, the present invention provides a milk exosome comprising:

a biological membrane surrounding a lumen;

a glycoprotein embedded within said biological membrane, wherein saidglycoprotein comprises a glycan present on the outer surface of saidbiological membrane; and

an exogenous cargo present in said lumen.

In another aspect, the present disclosure provides a milk exosomecomprising:

-   -   a biological membrane surrounding a lumen, wherein the        biological membrane comprises one or more glycoprotein(s),    -   wherein the biological membrane is modified as compared with the        natural biological membrane of the milk exosome.

In some embodiments, said exosome is isolated from sheep, goat, camel,horse, donkey, reindeer, yak, buffalo, or bovine (cow) milk orcolostrum.

In some embodiments, said exosome further comprises an miRNA or mRNAthat is biologically active in a mammal. In some embodiments, the miRNAor mRNA is present in the lumen of the exosome. In some embodiments, themiRNA or mRNA is endogenous miRNA or mRNA.

In some embodiments, said exogenous cargo is selected from one or morenucleic acid molecules, polypeptides, lipids, vitamins, minerals, smallmolecules, pharmaceuticals, hormones, or enzymes.

In some embodiments, said exogenous cargo comprises a therapeutic agent.In some embodiments, said therapeutic agent is selected from mRNAs,polypeptides, miRNAs, miRNA antagonists, nutrients, antibiotics, cancerdrugs, activators of Toll-like receptors, or molecules capable ofdelivery to macrophages. In some embodiments, said therapeutic agent isa cancer drug selected from a chemotherapeutic, an immunotherapeutic, ahormone therapeutic, or a targeted therapeutic.

In some embodiments, said exogenous cargo comprises a nutritional agent.In some embodiments, said nutritional agent is selected from vitamins,minerals, lipids, fatty acids, mRNAs, or polypeptides.

In some embodiments, said nutritional agent is a fatty acid selectedfrom omega-3 fatty acids or omega-6 fatty acids.

In another aspect, the present disclosure provides a milk exosomecomprising:

a biological membrane surrounding a lumen;

a glycoprotein embedded within said biological membrane, wherein saidglycoprotein comprises a modified glycan present on the outer surface ofsaid biological membrane; and

a cargo present in said lumen.

In some embodiments, said modified glycan modulates uptake of theexosome into a mammalian cell as compared with a corresponding glycanthat is naturally-occurring on said outer surface of said biologicalmembrane.

In some embodiments, said exosome is isolated from sheep, goat, camel,horse, donkey, reindeer, yak, buffalo, or bovine (cow) milk orcolostrum.

In some embodiments, the modified glycan modulates uptake into a humanreceptor cell.

In some embodiments, the receptor cell is selected from intestinalcells, venous endothelial cells or other endothelial cells, immunecells, macrophages, intestinal mucosa, peripheral cells of the liver,spleen, lung, brain, kidneys, or pancreas, cancer cells, or fetal cells.

In some embodiments, said modified glycan comprises one or more D- orL-glucose, erythrose, fucose, galactose, mannose, lyxose, gulose,xylose, arabinose, ribose, 2′-deoxyribose, glucosamine, lactosamine,polylactosamine, glucuronic acid, sialic acid, sialyl-Lewis X (SLex),N-acetyl-glucosamine, N-acetyl-galactosamine, neuraminic acid,N-glycolylneuraminic acid (Neu5Gc), N-acetylneuraminic acid (Neu5Ac), anN-glycan chain, an O-glycan chain, a Core 1, Core 2, Core 3, or Core 4structure, or a phosphate- or acetate-modified analog thereof or acombination thereof.

In some embodiments, said modified glycan reduces or eliminatesdegradation of the exosome by macrophages.

In some embodiments, the modified glycan is produced by removing one ormore glycans from the surface glycoproteins of a naturally-occurringmilk exosome and/or by removing the extrasomal surface portion of one ormore surface glycoproteins.

In some embodiments, the modified exosome is produced by contacting anaturally-occurring milk exosome with a chemical agent capable ofcleaving or covalently modifying glycans or proteins (e.g., hydrazine oran acylating or alkylating agent), or a protease or glycosidase orcombination thereof. In some embodiments, the exosome is produced bycontacting the naturally-occurring milk exosome with a lectin. In someembodiments, the exosome is produced by contacting thenaturally-occurring milk exosome with 3-N-acetylglucosaminidase, PNGaseA, PNGase F, Endoglycosidase H, Endoglycosidase F, β (1-4)Galactosidase, O-Glycosidase, a neuraminidase, Glu-C, Glc C, Asp-N,trypsin, and/or Arg-C; or any combination thereof. In some embodiments,the naturally-occurring milk exosome is contacted with PNGase, agalactosidase, O-glycosidase, O-glycosidase-N-acetyl-glucosamidase, or amixture thereof.

In some embodiments, the exosome is produced by introducing one or moreglycans to the surface glycoprotein.

In some embodiments, the exosome is produced using glycosylation thatadds one or more glycans to the surface glycoprotein.

In some embodiments, the exosome is produced by stabilizing one or moreglycans already present on the surface glycoprotein.

In some embodiments, the modified glycan improves stability of theexosome in the gastrointestinal tract, systemic circulation, lymphaticcirculation, intracellular conditions, or other tissues or organs of ahuman, for example, including without limitation liver, spleen, lung,brain, kidneys, or pancreas, cancer cells, or fetal cells.

In some embodiments, the modified glycan alters the stability of theexosome under physiological conditions in a human as compared with anexosome comprising the corresponding unmodified glycan.

In some embodiments, the modified glycan alters the stability of theexosome in the gastrointestinal tract, systemic circulation, lymphaticcirculation, or intracellular conditions of a human as compared with anexosome comprising the corresponding unmodified glycan.

In some embodiments, said cargo is selected from one or more nucleicacid molecules, polypeptides, lipids, vitamins, minerals, smallmolecules, pharmaceuticals, hormones, or enzymes.

In some embodiments, said cargo comprises a therapeutic agent. In someembodiments, said therapeutic agent is selected from mRNAs,polypeptides, miRNAs, miRNA antagonists, nutrients, antibiotics, cancerdrugs, activators of Toll-like receptors, or molecules capable ofdelivery to macrophages. In some embodiments, said therapeutic agent isa cancer drug selected from a chemotherapeutic, an immunotherapeutic, ahormone therapeutic, or a targeted therapeutic.

In some embodiments, said cargo comprises a nutritional agent. In someembodiments, said nutritional agent is selected from vitamins, minerals,lipids, fatty acids, mRNAs, or polypeptides. In some embodiments, saidnutritional agent is a fatty acid selected from omega-3 fatty acids oromega-6 fatty acids.

In another aspect, the present disclosure provides a nutritionalsupplement formulated for oral administration to a human, saidnutritional supplement comprising a disclosed exosome.

In some embodiments, the exosome comprises an endogenous cargo.

In some embodiments, the exosome comprises an exogenous cargo.

In another aspect, the present disclosure provides an infant formulacomprising a disclosed exosome or nutritional supplement. In someembodiments, the exosome comprises one or more nutritional agentsselected from vitamins, minerals, lipids, fatty acids, mRNAs, orpolypeptides.

In another aspect, the present disclosure provides a method of treatinga disease, disorder, or condition in a subject, comprising:

administering to said subject an effective amount of a disclosed exosomecomprising a cargo capable of treating said disease, disorder, orcondition.

In some embodiments, the disease, disorder, or condition is selectedfrom a hyperproliferative disorder, viral or microbial infection,autoimmune disease, allergic condition, inflammatory disease,cardiovascular disease, metabolic disease, or neurodegenerative disease.

In some embodiments, the disease, disorder, or condition is selectedfrom a proliferative disease. Exemplary proliferative diseases include abenign or malignant tumor, solid tumor, carcinoma of the brain, kidney,liver, adrenal gland, bladder, breast, stomach, gastric tumors, ovaries,colon, rectum, prostate, pancreas, lung, vagina, cervix, testis,genitourinary tract, esophagus, larynx, skin, bone or thyroid, sarcoma,glioblastomas, neuroblastomas, multiple myeloma, gastrointestinalcancer, colon carcinoma or colorectal adenoma, a tumor of the neck andhead, an epidermal hyperproliferation, psoriasis, prostate hyperplasia,a neoplasia, a neoplasia of epithelial character, adenoma,adenocarcinoma, keratoacanthoma, epidermoid carcinoma, large cellcarcinoma, non-small-cell lung carcinoma, lymphomas, Hodgkins andNon-Hodgkins lymphoma, a mammary carcinoma, follicular carcinoma,undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma,multiple myeloma, or a hematological malignancy (including leukemia,diffuse large B-cell lymphoma (DLBCL), ABC DLBCL, chronic lymphocyticleukemia (CLL), chronic lymphocytic lymphoma, primary effusion lymphoma,Burkitt lymphoma/leukemia, acute lymphocytic leukemia, B-cellprolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenström'smacroglobulinemia (WM), splenic marginal zone lymphoma, plasmacytoma,intravascular large B-cell lymphoma).

In some embodiments, the disease, disorder, or condition is selectedfrom inflammatory or obstructive airways diseases, resulting, forexample, in reduction of tissue damage, airways inflammation, bronchialhyperreactivity, remodeling or disease progression. Inflammatory orobstructive airways diseases to which the present invention isapplicable include asthma of whatever type or genesis including bothintrinsic (non-allergic) asthma and extrinsic (allergic) asthma, mildasthma, moderate asthma, severe asthma, bronchitic asthma,exercise-induced asthma, occupational asthma and asthma inducedfollowing bacterial infection. Treatment of asthma is also to beunderstood as embracing treatment of subjects, e.g. of less than 4 or 5years of age, exhibiting wheezing symptoms and diagnosed or diagnosableas “wheezy infants,” an established patient category of major medicalconcern and now often identified as incipient or early-phase asthmatics.

In some embodiments, the disease, disorder, or condition is selectedfrom heteroimmune diseases. Examples of such heteroimmune diseasesinclude, but are not limited to, graft versus host disease,transplantation, transfusion, anaphylaxis, allergies (e.g., allergies toplant pollens, latex, drugs, foods, insect poisons, animal hair, animaldander, dust mites, or cockroach calyx), type I hypersensitivity,allergic conjunctivitis, allergic rhinitis, and atopic dermatitis.

Other inflammatory or obstructive airways diseases and conditions towhich the present invention is applicable and include acute lung injury(ALI), adult/acute respiratory distress syndrome (ARDS), chronicobstructive pulmonary, airways or lung disease (COPD, COAD or COLD),including chronic bronchitis or dyspnea associated therewith, emphysema,as well as exacerbation of airways hyperreactivity consequent to otherdrug therapy, in particular other inhaled drug therapy. The invention isalso applicable to the treatment of bronchitis of whatever type orgenesis including, but not limited to, acute, arachidic, catarrhal,croupus, chronic or phthinoid bronchitis. Further inflammatory orobstructive airways diseases to which the present invention isapplicable include pneumoconiosis (an inflammatory, commonlyoccupational, disease of the lungs, frequently accompanied by airwaysobstruction, whether chronic or acute, and occasioned by repeatedinhalation of dusts) of whatever type or genesis, including, forexample, aluminosis, anthracosis, asbestosis, chalicosis, ptilosis,siderosis, silicosis, tabacosis and byssinosis.

In some embodiments, the disease, disorder, or condition is selectedfrom eosinophil related disorders, e.g. eosinophilia, in particulareosinophil related disorders of the airways (e.g. involving morbideosinophilic infiltration of pulmonary tissues) includinghypereosinophilia as it effects the airways and/or lungs as well as, forexample, eosinophil-related disorders of the airways consequential orconcomitant to Loffler's syndrome, eosinophilic pneumonia, parasitic (inparticular metazoan) infestation (including tropical eosinophilia),bronchopulmonary aspergillosis, polyarteritis nodosa (includingChurg-Strauss syndrome), eosinophilic granuloma and eosinophil-relateddisorders affecting the airways occasioned by drug-reaction.

In some embodiments, the disease, disorder, or condition is selectedfrom inflammatory or allergic conditions of the skin, for examplepsoriasis, contact dermatitis, atopic dermatitis, alopecia areata,erythema multiforma, dermatitis herpetiformis, scleroderma, vitiligo,hypersensitivity angiitis, urticaria, bullous pemphigoid, lupuserythematosus, systemic lupus erythematosus, pemphigus vulgaris,pemphigus foliaceus, paraneoplastic pemphigus, epidermolysis bullosaacquisita, acne vulgaris, and other inflammatory or allergic conditionsof the skin.

In some embodiments, the disease, disorder, or condition is selectedfrom diseases or conditions having an inflammatory component, forexample, treatment of diseases and conditions of the eye such as ocularallergy, conjunctivitis, keratoconjunctivitis sicca, and vernalconjunctivitis, diseases affecting the nose including allergic rhinitis,and inflammatory disease in which autoimmune reactions are implicated orhaving an autoimmune component or etiology, including autoimmunehematological disorders (e.g. hemolytic anemia, aplastic anemia, purered cell anemia and idiopathic thrombocytopenia), systemic lupuserythematosus, rheumatoid arthritis, polychondritis, scleroderma,Wegener granulamatosis, dermatomyositis, chronic active hepatitis,myasthenia gravis, Steven-Johnson syndrome, idiopathic sprue, autoimmuneinflammatory bowel disease (e.g. ulcerative colitis and Crohn'sdisease), irritable bowel syndrome, celiac disease, periodontitis,hyaline membrane disease, kidney disease, glomerular disease, alcoholicliver disease, multiple sclerosis, endocrine opthalmopathy, Grave'sdisease, sarcoidosis, alveolitis, chronic hypersensitivity pneumonitis,multiple sclerosis, primary biliary cirrhosis, uveitis (anterior andposterior), Sjogren's syndrome, keratoconjunctivitis sicca and vernalkeratoconjunctivitis, interstitial lung fibrosis, psoriatic arthritis,systemic juvenile idiopathic arthritis, cryopyrin-associated periodicsyndrome, nephritis, vasculitis, diverticulitis, interstitial cystitis,glomerulonephritis (with and without nephrotic syndrome, e.g. includingidiopathic nephrotic syndrome or minal change nephropathy), chronicgranulomatous disease, endometriosis, leptospiriosis renal disease,glaucoma, retinal disease, aging, headache, pain, complex regional painsyndrome, cardiac hypertrophy, musclewasting, catabolic disorders,obesity, fetal growth retardation, hyperchlolesterolemia, heart disease,chronic heart failure, mesothelioma, anhidrotic ecodermal dysplasia,Behcet's disease, incontinentia pigmenti, Paget's disease, pancreatitis,hereditary periodic fever syndrome, asthma (allergic and non-allergic,mild, moderate, severe, bronchitic, and exercise-induced), acute lunginjury, acute respiratory distress syndrome, eosinophilia,hypersensitivities, anaphylaxis, nasal sinusitis, ocular allergy, silicainduced diseases, COPD (reduction of damage, airways inflammation,bronchial hyperreactivity, remodeling or disease progression), pulmonarydisease, cystic fibrosis, acid-induced lung injury, pulmonaryhypertension, polyneuropathy, cataracts, muscle inflammation inconjunction with systemic sclerosis, inclusion body myositis, myastheniagravis, thyroiditis, Addison's disease, lichen planus, Type 1 diabetes,or Type 2 diabetes, appendicitis, atopic dermatitis, asthma, allergy,blepharitis, bronchiolitis, bronchitis, bursitis, cervicitis,cholangitis, cholecystitis, chronic graft rejection, colitis,conjunctivitis, Crohn's disease, cystitis, dacryoadenitis, dermatitis,dermatomyositis, encephalitis, endocarditis, endometritis, enteritis,enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis,gastritis, gastroenteritis, Henoch-Schonlein purpura, hepatitis,hidradenitis suppurativa, immunoglobulin A nephropathy, interstitiallung disease, laryngitis, mastitis, meningitis, myelitis myocarditis,myositis, nephritis, oophoritis, orchitis, osteitis, otitis,pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis,pleuritis, phlebitis, pneumonitis, pneumonia, polymyositis, proctitis,prostatitis, pyelonephritis, rhinitis, salpingitis, sinusitis,stomatitis, synovitis, tendonitis, tonsillitis, ulcerative colitis,uveitis, vaginitis, vasculitis, or vulvitis.

In some embodiments the inflammatory disease which can be treatedaccording to the methods of this invention is an disease of the skin. Insome embodiments, the inflammatory disease of the skin is selected fromcontact dermatitits, atompic dermatitis, alopecia areata, erythemamultiforma, dermatitis herpetiformis, scleroderma, vitiligo,hypersensitivity angiitis, urticaria, bullous pemphigoid, pemphigusvulgaris, pemphigus foliaceus, paraneoplastic pemphigus, epidermolysisbullosa acquisita, and other inflammatory or allergic conditions of theskin.

In some embodiments the inflammatory disease which can be treatedaccording to the methods of this invention is selected from acute andchronic gout, chronic gouty arthritis, psoriasis, psoriatic arthritis,rheumatoid arthritis, Juvenile rheumatoid arthritis, Systemic jubenileidiopathic arthritis (SJIA), Cryopyrin Associated Periodic Syndrome(CAPS), and osteoarthritis.

In some embodiments the inflammatory disease which can be treatedaccording to the methods of this invention is a TH17 mediated disease.In some embodiments the TH17 mediated disease is selected from Systemiclupus erythematosus, Multiple sclerosis, and inflammatory bowel disease(including Crohn's disease or ulcerative colitis).

In some embodiments the inflammatory disease which can be treatedaccording to the methods of this invention is selected from Sjogren'ssyndrome, allergic disorders, osteoarthritis, conditions of the eye suchas ocular allergy, conjunctivitis, keratoconjunctivitis sicca and vernalconjunctivitis, and diseases affecting the nose such as allergicrhinitis.

Cardiovascular diseases which can be treated according to the methods ofthis invention include, but are not limited to, restenosis,cardiomegaly, atherosclerosis, myocardial infarction, ischemic stroke,congestive heart failure, angina pectoris, reocclusion afterangioplasty, restenosis after angioplasty, reocclusion afteraortocoronary bypass, restenosis after aortocoronary bypass, stroke,transitory ischemia, a peripheral arterial occlusive disorder, pulmonaryembolism, and deep venous thrombosis.

In some embodiments, the neurodegenerative disease which can be treatedaccording to the methods of this invention include, but are not limitedto, Alzheimer's disease, Parkinson's disease, amyotrophic lateralsclerosis, Huntington's disease, cerebral ischemia, andneurodegenerative disease caused by traumatic injury, glutamateneurotoxicity, hypoxia, epilepsy, treatment of diabetes, metabolicsyndrome, obesity, organ transplantation and graft versus host disease.

In another aspect, the present disclosure provides a method ofcorrecting dysbiosis or improving the gut microbiome or gut health of asubject, comprising:

administering to said subject an effective amount of a disclosed exosomecomprising a cargo;

wherein the cargo of said exosome is effective to correct dysbiosis orimprove the gut microbiome or gut health of said subject.

In some embodiments, said correcting dysbiosis or improving the gutmicrobiome or gut health comprises a decrease in Ruminococcaceae and/orVerrucomicrobiae.

In some embodiments, said correcting dysbiosis or improving the gutmicrobiome or gut health comprises an increase in Clostridiales orErysipelotrichaceae.

In another aspect, the present disclosure provides a method of treatinginflammatory bowel disease, obesity, or non-alcoholic fatty liverdisease, comprising: administering to a subject in need thereof aneffective amount of a disclosed exosome, wherein the exosome comprises acargo capable of treating inflammatory bowel disease, obesity, ornon-alcoholic fatty liver disease.

In another aspect, the present disclosure provides a method ofincreasing muscle strength, enhancing sensorimotor gating or cognitiveperformance, or increasing fertility or fecundity in a subject,comprising administering to said subject an effective amount of adisclosed exosome or a disclosed nutritional supplement.

In another aspect, the present disclosure provides a method of treatingsarcopenia, muscle loss after injury, atherosclerosis, cancer, an immunedisease, impaired fecundity, or cognitive impairment, comprisingadministering to a subject in need thereof a disclosed exosome or adisclosed nutritional supplement.

In another aspect, the present disclosure provides a method of improvingstability or uptake selectivity of a milk exosome in thegastrointestinal tract, systemic circulation, lymphatic circulation,intracellular conditions, or other tissue or organ of a human,comprising:

i) providing a disclosed milk exosome; and

ii) altering the exosome of step i) by: removing one or more glycansfrom a surface glycoprotein of the exosome; removing an extrasomalsurface portion of the surface glycoprotein; introducing one or moreglycans to a surface glycoprotein; stabilizing one or more glycansalready present on a surface glycoprotein; or a combination thereof.

In some embodiments, step ii) comprises contacting the exosome of stepi) with β-N-acetylglucosaminidase, PNGase A, PNGase F, EndoglycosidaseH, Endoglycosidase F, β (1-4) Galactosidase, O-Glycosidase, aneuraminidase, Glu-C, Glc C, Asp-N, trypsin, and/or Arg-C; or anycombination thereof.

In some embodiments, the method promotes selective delivery of the milkexosome or its cargo to cells in the gastrointestinal tract as comparedwith a milk exosome comprising the corresponding unmodified glycan. Insome embodiments, the method promotes selective delivery of the milkexosome or its cargo to cells in the systemic circulation as comparedwith a milk exosome comprising the corresponding unmodified glycan. Insome embodiments, the method promotes selective delivery of the milkexosome or its cargo to cells in the lymphatic circulation as comparedwith a milk exosome comprising the corresponding unmodified glycan. Insome embodiments, the method promotes selective delivery of the milkexosome or its cargo to cells in the liver, spleen, lung, brain,kidneys, pancreas, cancer cell, or fetal cell as compared with a milkexosome comprising the corresponding unmodified glycan.

In some embodiments, said subject is a human.

Extracellular Vesicles

Extracellular vesicles that can be used to encapsulate or carry one ormore cargos as described herein include a biological membrane (e.g., alipid bilayer) that surrounds a lumen. Any appropriate extracellularvesicle can be used as described herein. Examples of extracellularvesicles include, without limitation, exosomes, microvesicles,oncosomes, ectosomes, prostasomes, matrix/calcifying vesicles,tolerosomes, cardiosomes, and vexosomes. Extracellular vesicles andtheir respective properties are discussed elsewhere (see, e.g., Lotvallet al. 2014 Journal of Extracellular Vesicles 3:26913; and Zempleni etal., 2013 Nature Reviews Drug Discovery 12:347-357). In someembodiments, an exosome can be used to carry or encapsulate one or morecargos as described herein. In some embodiments, an exosome comprises abiological membrane surrounding a lumen, glycoprotein(s) embedded withinthe biological membrane such that one or more glycans on theglycoprotein(s) are presented on the outer surface of the biologicalmembrane, and cargo encapsulated in the lumen.

An extracellular vesicle described herein can be obtained by anyappropriate method. In embodiments where an extracellular vesicle is anexosome, the exosome can be a milk exosome. As used herein a “milkexosome” is any exosome found in the milk or colostrum of a mammal(e.g., a lactating mammal), such as sheep, goat, camel, horse, donkey,reindeer, yak, buffalo, or bovine (cow) milk or colostrum. In someembodiments, an exosome is isolated from milk. A milk exosome can beisolated from milk by, for example, centrifugation (e.g.,ultracentrifugation), size exclusion chromatography, affinitychromatography, and density gradient centrifugation. In someembodiments, milk exosomes isolated from milk can be concentrated,purified, and/or modified (e.g., loaded with one or more cargos). A milkexosome can be obtained from any appropriate mammal, including humans.Examples of non-human mammals include, without limitation, non-humanprimates (such as monkeys), cows, pigs, goats, horses, and donkeys. Insome embodiments, an extracellular vesicle that can be used as describedherein is a bovine milk exosome. In other embodiments, an extracellularvesicle that can be used as described herein is a goat milk exosome. Inother embodiments, an extracellular vesicle that can be used asdescribed herein is a pig milk exosome.

Methods for isolating a milk exosome as well as methods for determiningpurity thereof are provided in the Examples section.

Biological Membranes of the Extracellular Vesicles

Glycoproteins belong to a class of proteins having one or morecarbohydrate groups attached to the polypeptide chain. Glycoproteinscontain oligosaccharide chains (glycans) covalently attached topolypeptide side-chains. The carbohydrate is attached to the protein ina cotranslational or posttranslational modification process known asglycosylation. Glycoproteins are found on the outside biologicalmembranes, with the sugar facing out. Thus, glycoproteins are often animportant integral membrane proteins, where they play a role incell-cell interactions. There are several types of glycosylation:N-glycosylation in which sugars are attached to nitrogen, typically onthe amide side-chain of asparagine through N-glycosidic bonds;O-glycosylation in which sugars are attached to oxygen, typically onserine or threonine but also on non-canonical amino acids such ashydroxylysine & hydroxyproline through O-glycosidic bonds;P-glycosylationin which sugars are attached to phosphorus on aphosphoserine; C-glycosylation in which sugars are attached directly tocarbon, such as in the addition of mannose to tryptophan. The differentstructure of N- and O-linked sugars give them different functions.

The eight sugars typically found in eukaryotic glycoproteins includeβ-D-Glucose (Glc) which is a hexose sugar, β-D-Galactose (Gal) which isa hexose sugar, β-D-Mannose (Man) which is a hexose sugar,N-Acetylneuraminic acid (NeuNAc) which is a Sialic acid, α-L-Fucose(Fuc) which is a deoxyhexose sugar, N-Acetylgalactosamine (GalNAc) whichis an amino hexose, N-Acetylglucosamine (GcNAc) which is an amino hexoseXylose (Xyl) which is a pentose. The sugar group(s) can assist inprotein folding or improve a protein's stability. Examples ofglycoproteins include lectins, mucins, and several polypeptide hormones.Glycoproteins are found on the outside biological membranes, with thesugar facing out. Thus, glycoproteins are often an important integralmembrane proteins, where they play a role in cell-cell interactions.

An extracellular vesicle, e.g., exosome, that can be used to encapsulateone or more cargos as described herein can include a biological membranecontaining one or more glycoproteins. An extracellular vesicle, e.g.,exosome, that can be used to carry one or more cargos as describedherein can include a biological membrane containing one or moreglycoproteins. In some embodiments, an extracellular vesicle describedherein can include a biological membrane comprising one or more surfaceglycoproteins. For example, one or more glycoproteins can be embeddedwithin or otherwise present in or present on the surface of thebiological membrane of an extracellular vesicle such that a glycan onthe glycoprotein is presented on the outer surface of the biologicalmembrane and thus the outer surface of the extracellular vesicle.

Glycoproteins that can be present in the biological membrane of anextracellular vesicle as described herein can include any appropriateglycan. Examples of glycans include, without limitation, N-glycans(e.g., N-acetyl-glucosamines and N-glycan chains), O-glycans, C-glycans,sialic acid, galactose or mannose residues, and combinations thereof. Insome embodiments, the glycan is selected from an alpha-linked mannose,Gal β 1-3 GaNAc 1 Ser/Thr, GalNAc, or sialic acid. In any of theseembodiments, the extracellular vesicle is an exosome, e.g., a milkexosome.

In some embodiments, an extracellular vesicle that can be used to carryor encapsulate one or more cargos as described herein can include one ormore modified glycoproteins. In some embodiments, the extracellularvesicle comprising one or more modified glycoproteins is an exosome,e.g., a milk exosome. Cellular uptake of milk exosomes (e.g., bovinemilk exosome) is mediated by endocytosis and depends on both cell andexosome surface glycoproteins (see, e.g., Wolf et al., 2015 Journal ofNutrition 145:2201-2216). Modification the glycan(s) presented on theouter surface of the exosome can be used to control or alter cellularuptake of extracellular vesicles and/or delivery of cargo present in theexosome.

In some embodiments, the extracellular vesicle, e.g., exosome, can havea modified biological membrane such that one or more of its nativeglycoproteins is increased, decreased or altered. Thus in someembodiments, the present disclosure provides an extracellular vesicle,e,g., an exosome (e.g., milk exosome) comprising a biological membranesurrounding a lumen, wherein the biological membrane comprises one ormore glycoprotein(s), wherein the biological membrane is modified ascompared with the natural biological membrane of the milk exosome. Insome embodiments, the extracellular vesicle (EV) or exosome contains abiological membrane which is modified such that it has an increasednumber of one or more of its native glycoprotein(s). In someembodiments, the EV or exosome contains a biological membrane ismodified such that it has a decreased number of one or more of itsnative glycoprotein(s). In some embodiments, the EV or exosome containsa biological membrane which is modified such that one or more of itsnative glycoprotein(s) is not present. In some embodiments, the EV orexosome contains a biological membrane that is modified such that itincludes one or more glycoprotein(s) that is not naturally present inthe natural biological membrane. In some embodiments, the EV or exosomecontains a biological membrane that is modified such that one or more ofits native glycoprotein(s) is altered.

The biological membrane of the EV or exosome can be modified to increaseone or more native glycoproteins or can be modified to includeglycoproteins that are not naturally present in the native biologicalmembrane using various methods known in the art to deliver or embedglycoproteins into the membranes of EV or exosomes, including forexample, electroporation or transfection with cationic lipid reagents.Other methods include delivering the glycoprotein(s) byultracentrifugation including using methods described in U.S. Pat. No.9,085,778, US 2016/0000710, and WO 2015/161184, each of which is herebyincorporated by reference, as well as methods described in Luan, X. etal., Acta Pharmacol Sin. 2017 June; 38(6): 754-763 and Munagala R, etal., Cancer Lett. 2016 Feb. 1; 371(1):48-61, each of which is herebyincorporated by reference. Those procedures include i) suspending theglycoprotein(s) in PEG-400, mixing with milk-derived exosomes, followedby low-speed centrifugation; ii) mixing with milk- or colostrum-derivedexosomes, low-speed centrifugation (10,000×g) to remove any glycoproteinthat did not get incorporated into the biological membrane, and finallyhigh-speed centrifugation; and iii) mixing the glycoprotein(s) inethanol with 100,000 whey (obtained after the 100,000×g centrifugation),low-speed centrifugation and finally 120,000×g centrifugation. There aremany methods, such as the methods described in the Examples section thatcan be used to confirm that the added glycoprotein(s) are present in thebiological membrane of the EV or exosome.

The biological membrane of the EV or exosome can be modified to decreaseor remove one or more native glycoproteins by contacting the EV orexosome with one or more proteases, which cleave amino acids. Thus, insome embodiments, the EV or exosome is produced by contacting it with anenzyme selected from a serine protease, cysteine protease ormetalloprotease. In some embodiments, the EV or exosome is produced bycontacting it with an enzyme is selected from trypsin, AspN, GluC, ArgC,chymotrypsin, proteinase K, and Lys-C. The specificities of thesedifferent proteases are provided herein in Table A. Methods for treatingEV or exosomes with such proteases to decrease or remove glycoprotein(s)from the biological membrane of EV or exosomes are provided in detail inthe Examples section.

Glycoproteins and Glycans

As with other major classes of macromolecules, the biological roles ofglycans span the spectrum from those that appear to be relativelysubtle, to those that are crucial for the development, growth,functioning, or survival of the organism that synthesizes them. See,e.g., Essentials of Glycobiology, 2nd edition, Varki, A, Cummings, R.D., Esko, J. D., et al., eds. Cold Spring Harbor (N.Y.): Cold SpringHarbor Laboratory Press; 2009.

The biological roles of glycans can be divided into two broadcategories: (1) the structural and modulatory properties of glycans and(2) the specific recognition of glycans by other molecules-mostcommonly, glycan-binding proteins (GBPs). The GBPs can be subdividedinto two major groups: (1) intrinsic GBPs, which recognize glycans fromthe same organism and (2) extrinsic GBPs, which recognize glycans from adifferent organism. Intrinsic GBPs typically mediate cell-cellinteractions or recognize extracellular molecules, but they can alsorecognize glycans on the same cell. Extrinsic GBPs consist mostly ofpathogenic microbial adhesins, agglutinins, or toxins, but some alsomediate symbiotic relationships. These two types of glycan recognitionlikely act as opposing selective forces driving evolutionary change, atleast partly accounting for the enormous diversity of glycan structurefound in nature. Further complexity arises from the fact that somemicrobial pathogens engage in “molecular mimicry,” evading immunereactions by decorating themselves with glycans typical of their hosts.Finally, some microbes are themselves targets of their own pathogens(e.g., bacteriophages that invade bacteria), and glycan recognition is acommon feature of these interactions as well.

Approaches taken to understand the biological roles of glycans includethe prevention of initial glycosylation, prevention of glycan chainelongation, alteration of glycan processing, enzymatic or chemicaldeglycosylation of completed chains, genetic elimination ofglycosylation sites, and the study of naturally occurring geneticvariants and mutants in glycosylation.

The first intrinsic glycan receptors to be identified were those thatmediate clearance, turnover, and intracellular trafficking of solubleblood-plasma glycoproteins. Most of these receptors specificallyrecognize certain terminal or subterminal glycans on the solubleglycoprotein. There are also endocytic receptors, whose functions haveyet to be assigned, that recognize specific glycan sequences. Severalinstances exist wherein free glycans can have hormonal actions thatinduce specific responses in a highly structure-specific manner.Examples include the interaction of small glycans from bacterialsymbionts with plant roots and the bioactive properties of fragments ofhyaluronan in mammalian systems, both of which can induce biologicalresponses in a size- and structure-dependent manner. Likewise, freeheparan or dermatan sulfate fragments released by certain cell types canhave major biological effects in complex situations such as woundhealing.

It is now clear that glycans have many specific biological roles incell-cell recognition and cell-matrix interactions. One of the bestcharacterized examples concerns the selectin family of adhesionmolecules, which recognize glycan structures on their ligands andthereby mediate critical interactions between blood cells and vascularcells in a wide variety of normal and pathological situations. Asindicated above, GBPs and glycans present on cell surfaces can interactspecifically with molecules in the matrix or even with glycans on thesame cell surface. In some such instances, the specific biologicalsignificance of recognition has yet to be conclusively demonstrated inthe intact animal. Also, it is becoming clear that some criticalrecognition sites are actually combinations of glycans and protein. Forexample, P-selectin recognizes the generic selectin ligand sialyl Lewisxwith high affinity only in the context of the amino-terminal 13 aminoacids of P-selectin glycoprotein ligand-1 (PSGL-1), which includecertain required sulfated tyrosine residues. More recently, a differentform of intrinsic recognition has been described, in whichglycan-binding sites of cell-surface receptors are masked by cognateglycans on the same cell surface, making them unavailable forrecognition by external ligands. Generally speaking, terminal sugarsequences, unusual structures, or modifications of the glycans are morelikely to be involved in recognizing highly specific cell types or otherparticular, unique recognition events.

Carbohydrate-carbohydrate interactions may also have a specific role incell-cell interactions and adhesion. A dramatic example is thespecies-specific interaction between marine sponges, which is mediatedvia homotypic binding of the glycans on a large cell-surfaceglycoprotein. Certain glycans act as specific binding sites for avariety of viruses, bacteria, and parasites, and as recognition targetsfor many plant and bacterial toxins. In such situations, there istypically excellent recognition specificity for the sequence of theglycan involved. For example, the hemagglutinins of many virusesspecifically recognize the type of host sialic acid, its modifications,and its linkage to the underlying sugar chain. Likewise, various toxinsbind with high specificity to certain gangliosides but not to relatedstructures. There is little doubt about the importance of structuralspecificity with respect to these functions of glycans. Indeed, many ofthe microbial binding proteins involved have been harnessed as specifictools for studying the expression of the cognate sugar chains.

Glycans can mediate uptake by and interactions with specific cell types,for example immune cells. Antigenic proteins must first be taken up byantigen presenting cells (macrophages and especially dendritic cells),which process them into peptides, to be presented by MHC Class IImolecules, for recognition by T lymphocytes. This process can befacilitated by glycans on the target protein. For example, the presenceof high densities of terminal Man or GlcNAc residues on foreign proteinsor microbes can trigger phagocytosis via C-type lectins on antigenpresenting cells, with resulting delivery of the antigenic proteins toprocessing compartments.

A variety of cell surface receptors that recognize terminal glycans cantrigger uptake of molecules (endocytosis), particles (phagocytosis) oreven intact cells. The classic examples are the asialoglycoproteinreceptor of hepatocytes and the mannose receptor of macrophages, whichbind to mannose glycans and mannose 6-phosphate glycans. The mannosereceptor (Cluster of Differentiation 206, CD206) is a C-type lectinprimarily present on the surface of macrophages and immature dendriticcells, but is also expressed on the surface of skin cells such as humandermal fibroblasts and keratinocytes. A C-type lectin (CLEC) is a typeof carbohydrate-binding protein domain known as a lectin. The C-typedesignation is from their requirement for calcium for binding. Proteinsthat contain C-type lectin domains have a diverse range of functionsincluding cell-cell adhesion, immune response to pathogens andapoptosis. Another glycan receptor is the sialic acid receptorSialoadhesin which is expressed by macrophage subsets. A large varietyof lectins are known to carry out endocytosis in macrophages anddendritic cells. Such recognition processes may be critical not only forproviding antigens to process and present to T cells, but also forclearing away damaged cells or glycoproteins, such as occurs whenmicrobial sialidases enter the circulation during sepsis and causedesialylation of platelets, or when cancers secrete incompletelyglycosylated mucins.

Lectins

Antiglycan antibodies and lectins are widely used in glycan analysisbecause their specificities enable them to discriminate among a varietyof glycan structures and their multivalency ensures high-affinitybinding to the glycans and cell surfaces containing those glycans.

Many of the lectins currently used as tools in glycobiology come fromplants and are commercially available. Most of these lectins werecharacterized initially by inhibition assays, in which monosaccharides,monosaccharide derivatives, or small oligosaccharides are used to blocklectin binding to cells or some other glycan-coated target. Smallmolecules that compete with binding of a lectin or antibody to alarger-sized ligand are termed haptens. These lectins are grouped byspecificity depending on the monosaccharide(s) for which they show thehighest affinity and their distinct preference for α- or β-anomers ofthe sugar. However, lectins within a particular specificity group alsomay differ in their affinities for different glycans. The common methodfor grouping lectins according to monosaccharide specificity should thusbe used with caution because it does not reflect the complex specificdeterminants a given lectin may recognize with high affinity. Thebinding affinity (K_(d)) of lectins for complex glycans is often in therange of 1 to 10 μM. For complex glycoconjugates with multipledeterminants or multivalency, the binding affinity of lectins mayapproach nanomolar values. In contrast, the affinity of most lectins formonosaccharides is in the millimolar range. The specificity ofconcanavalin A (ConA), perhaps the most widely used lectin, demonstratesthis point. This lectin (which is an α-mannose/α-glucose-binding lectin)binds to N-glycans and is not known to bind O-glycans on animal cellglycoproteins. However, it binds oligomannose-type N-glycans with muchhigher affinity than it binds complex-type biantennary N-glycans, and itfails to bind more highly branched complex-type N-glycans.

Lectins and antibodies are useful reagents for aiding in glycanidentification. See, e.g., Essentials of Glycobiology, [internet] 3rdedition, Varki, A, Cummings, R. D., Esko, J. D., et al., eds. ColdSpring Harbor (N.Y.): Cold Spring Harbor Laboratory Press; 2015-2017.They include agglutination of cells and blood typing, cell separationand analysis, bacterial typing, identification and selection of mutatedcells with altered glycosylation, toxic conjugates for tumor cellkilling, cytochemical characterization/staining of cells and tissues,inducing mitogenesis of cells, acting as growth inhibitors, mappingneuronal pathways, purification and characterization ofglyco-conjugates, assays of glycosyltransferases and glycosidases, anddefining glycosylation status of target glycoconjugates and cells. Thus,using a variety of lectins and antibodies, it is possible to deduce manyaspects of glycan structures. Microarrays in which a variety of lectinsand antibodies are printed on a slide can also give valuable informationabout the glycosylation status of cells and glycoconjugates. Thisapproach is especially sensitive in regard to defining whetherbiological samples differ in glycosylation. For example, such approacheshave been adapted to study differential glycosylation of prionglycoproteins using a panel of biotinylated lectins in ELISA-typeformats.

Exosome Glycoproteins and Effects on Uptake and Delivery

Exosomes secreted from cells intrinsically express some lipids and celladhesion molecules and ligands that naturally target certain types ofrecipient cells. Several studies have shown that cell-secreted exosomeshave natural targeting ability based on their donor cells of origin(Luan, X. et al., “Engineering exosomes as refined biologicalnanoplatforms for drug delivery,” Acta Pharmacologica Sinica 2017, 38:754-763, hereby incorporated by reference). For instance, exosomesisolated from neuroblastoma intrinsically express glycosphingolipidglycan groups that can bind to the aggregates of amyloid-β in the brain,and therefore may provide an effective treatment for Alzheimer's disease(see, e.g., Hood, J. L., “Post isolation modification of exosomes fornanomedicine applications,” Nanomedicine (Lond) 2016, 11, 1745-56,hereby incorporated by reference). As demonstrated by the presentdisclosure, it has now been found that modulation of the targeting anduptake of milk exosomes is possible. Methods of characterizing andaltering surface glycoproteins and glycans are known in the art. Forexample, after cleavage of surface peptides with one or more proteases,the peptides may be identified using LC/MS-MS, Mascot and Sequestdatabases. Glycoproteins may be identified using a series of tools basedon neuronal networks including NetNglyc, NetOglyc, NetCglyc 1.0 andGlycoEP, and the like.

Targeting ligands on the surfaces of some cell-secreted exosomes can beengineered. The most commonly used technique is to insert the geneencoding the targeting proteins into the donor cells that produce theexosomes of interest. The donor cells then secrete this protein in theexosomes. For example, plasmids encoding Lamp2b have been constructedand transfected into dendritic cells. The exosomes harvested after thedonor cells have been transfected and found to fuse strongly to theneuron-specific rabies viral glycoprotein (RVG) peptide through Lamp2bon the exosomal membrane. The expression of Lamp2b on the exosomes hasbeen confirmed by western blotting. These targeted exosomes caneffectively deliver siRNA to the brain in a mouse model (see, e.g.,Alvarez-Erviti L. et al., “Delivery of siRNA to the mouse brain bysystemic injection of targeted exosomes,” Nat Biotechnol 2011, 29,341-5, hereby incorporated by reference). Another study has usedexosomes to deliver let-7a miRNA in a targeted manner toEGFR-overexpressing breast cancer cells in mice. GE11 or EGF was clonedinto a pDisplay vector and transfected into HEK299 cells. The datasuggest that intravenous injection of the let-7α-loaded GE11-targetingexosomes can deliver the gene to the EGFR-expressing tumor in a mousexenograft model (Ohno, S. et al., “Systemically injected exosomestargeted to EGFR deliver antitumor microRNA to breast cancer cells,” MolTher 2013, 21, 185-91, hereby incorporated by reference).

As disclosed herein, surface glycoproteins and their specific surfaceglycans affect the uptake and delivery of microvesicles such as milkexosomes (Sukreet, S., et al., “Identification of Glycoproteins on theSurface of Bovine Milk Exosomes and Intestinal Cells that FacilitateExosome Uptake in Human Colon Carcinoma Caco-2 Cells,” The FASEB Journal2017, 31, 646.25, hereby incorporated by reference). Exosomes wereisolated from bovine milk by differential centrifugation andauthenticated as recommended by International Society for ExtracellularVesicles. Surface proteins or glycan modifications in exosomes or Caco-2cells were removed using proteases (Glu-C, trypsin, Arg-C, Asp-N, orproteinase K) or glycosidases [N-glycosidase F (PNGase F),galactosidase, O-glycosidase, neuraminidase, N-acetyl glucosaminidase,or a combination of all]. Controls were incubated with solvent. Exosomeswere labeled with FM4-64, and unlabeled fluorophore was removed. Surfacepeptides released after the treatment were identified using LC/MS-MS,Mascot and Sequest databases. Glycoproteins on the external surface wereidentified using a series of tools based on neuronal networks includingNetNglyc, NetOglyc, NetCglyc 1.0 and GlycoEP. Transport kinetics weremodelled using the Michaelis-Menten equation. One-way ANOVA andBonferroni's multiple comparison were used for statistical analyses.Treatment with galactosidase, PNGase F and O-glycosidase had a strongereffect than other glycosidases. When exosomes or cells were treated withthe glycosidase mixture, no uptake was detected or uptake was decreasedby 80%, respectively, in comparison to controls. 149 total proteins wereidentified in bovine exosome, including 4 (N), 2 (O) and 2 (C) glycanbinding sites; 298 proteins were identified in Caco-2 cells, including46 membrane proteins that included 29 (N), 33 (O) and 6 (C) glycanbinding sites. Thus, glycoproteins on the surface of bovine milkexosomes and intestinal cells facilitate the uptake of exosomes in humanintestinal cells. The β-galactoside, Core 1 & Core 3-O-linkeddisaccharide and N-acetylglucosamine modifications appear to be ofgreater importance than other glycan features present on complexglycoproteins.

In some embodiments, one or more glycan(s) present on the outer surfaceof the extracellular vesicle is modified by alteration, substitution,addition, and/or deletion of one or more amino acid residues. In someembodiments, the biological membrane of the EV or exosome is modifiedsuch that one or more of its native glycoprotein(s) is altered. In someembodiments, the one or more native glycoprotein(s) is altered such thatthe number of glycan residues present on the glycoprotein(s) isincreased. In some embodiments, the exosome is produced usingglycosylation that adds one or more glycans to the glycoprotein. In someembodiments, the one or more native glycoprotein(s) is altered such thatthe number of glycan residues present on the glycoprotein(s) isdecreased. In some embodiments, the number of glycan residues isdecreased by cleavage of one or more glycan residues present on theglycoprotein(s). In some embodiments, the exosome is produced using anenzyme selected from a glycosidase, exoglycosidase, endoglycosidase,glycoamidase, neuraminidase, galactosidase, peptide:N-glycosidase(PNGase), glycohydrolase, and any combination thereof wherein the EV orexosome is contacted with the enzyme to remove one or more glycans. Insome embodiments, the enzyme is selected from aβ-N-acetylglucosaminidase, PNGase F, β (1-4) Galactosidase,O-Glycosidase, N-Glycosidase, N-glycohydrolase, Endo H, Endo D, Endo F₂,EndoF₃, and any combination thereof.

In some embodiments, two or more native glycoprotein(s) are altered suchthat at least one glycoprotein has an increased number of glycanresidues and at least one other glycoprotein has a decreased number ofglycan residues or is missing its glycan residue(s), wherein theglycoprotein(s) having an increased number of glycan residues isdifferent from the glycoprotein(s) having a decreased number of glycanresidues or missing glycan residues. In some embodiments, the one ormore native glycoprotein(s) is altered such that it comprises a modifiedglycan. In some embodiments, the modified glycan comprises at least onecarbohydrate moiety that differs from that of the glycan in the nativeglycoprotein(s). In some embodiments, the modified glycan comprises oneor more galactose, mannose, glucose, O-glycans, N-acetyl-glucosamine,sialic acid, xylose, fucose, and/or N-glycan chains or any combinationthereof. In some embodiments, the modified glycan lacks a portion of oneor more of its carbohydrate chain(s). In some embodiments, the modifiedglycan is missing one or more of its carbohydrate chain(s). In someembodiments, the modified glycan comprises one or more alteredcarbohydrate chain(s). In some embodiments, the one or more nativeglycoprotein(s) is altered such that at least one glycan present on theglycoprotein(s) is substituted with a glycan that is not naturallypresent in the native glycoprotein(s). In some embodiments, the one ormore native glycoprotein(s) is altered by blocking one or more glycanresidue(s) present on the glycoprotein(s). In some embodiments, the oneor more glycan residue(s) is blocked by lectin binding to the glycanresidue. In some embodiments the glycan is blocked by an antibody thatbinds to that glycan.

In some embodiments, modification of the glycan(s) present on the outersurface of the exosome is used to target the exosome to specific celltypes. For example, polypeptide sequences adjacent to arginine andlysine residues in glycans are essential for exosome uptake bymacrophages. In some embodiments, modification the glycan(s) presentedon the outer surface of the exosome can be used to reduce and/oreliminate degradation of the exosome. In some embodiments, modification,e.g., alteration, substitution, addition, and/or deletion, ofpolypeptide sequences adjacent to arginine and lysine residues in aglycan(s) present on the outer surface of an exosome is used to reduceand/or eliminate degradation of an exosome (e.g., by macrophages). Insome embodiments, removal of galactose residues in a glycan(s) presenton the outer surface of an exosome is used to decrease the uptake of theexosome into intestinal cells.

In some embodiments, alteration or substitution of the glycan comprisesadding a glucose, fucose, neuraminic acid, allose, xylose, ribose,arabinose, threose, galactose, mannose, or sialic acid moiety. In someembodiments, alteration or substitution of the glycan comprises addingan alpha-linked mannose, Gal β 1-3 GalNAc 1 Ser/Thr, GalNAc, or sialicacid.

An extracellular vesicle having one or more modified glycoproteinsdescribed herein can be made using any appropriate method. In someembodiments, an extracellular vesicle having one or more modifiedglycoproteins described herein can be made by removing certain glycansfrom the one or more surface glycoproteins and/or by removing theextrasomal surface portion of a surface glycoprotein. For example, anexosome can be treated with a protease (e.g., Glu-C (targeting glu),trypsin (targeting arg and lys), and/or Arg-C (targeting arg and lys))to remove the extrasomal surface portion of a surface glycoprotein. Insome embodiments, an EV or exosome can be contacted with a glycosidaseto remove one or more glycan residues, for example, an enzyme selectedfrom a glycosidase, exoglycosidase, endoglycosidase, glycoamidase,neuraminidase, galactosidase, peptide:N-glycosidase (PNGase),glycohydrolase, and any combination thereof. Specific exemplary enzymesinclude β-N-acetylglucosaminidase, PNGase F, β (1-4) Galactosidase,O-Glycosidase, N-Glycosidase, N-glycohydrolase, Endo H, Endo D, Endo F₂,EndoF₃, and any combination thereof which can be used to remove one ormore glycans from the EV or exosomes. Methods for contacting exosomeswith such enzymes are provided in the Examples section. In anotherembodiment, EVs or exosomes have glycoproteins with one or more modifiedglycans can be produced using a transgenic animal that has one or moreglycan transferase enzyme(s) knocked out. Exemplary methods are providein Example 9. In other embodiments, one or more native glycoprotein(s)is altered by blocking one or more glycan residue(s) present on theglycoprotein(s). In some embodiments, one or more glycan residue(s) isblocked by lectin binding to the glycan residue. In some embodiments,the lectin is selected from Concanavalin A, Lentil lectin, Snowdroplectin, Ricin (Ricinus communis Agglutinin, RCA120), Peanut agglutinin,Jacalin, Hairy vetch lectin, Dolichos biflorus agglutinin, Soybeanagglutinin, N-acetylglucosamine binding lectins, Wheat Germ Agglutinin(WGA), Phaseolus vulgaris agglutinin, Elderberry lectin, Maackiaamurensis leukoagglutinin, Maackia amurensis hemoagglutinin, Ulexeuropaeus agglutinin, or Aleuria aurantia lectin.

In some embodiments, an extracellular vesicle having one or moremodified glycoproteins described herein can be made by introducing oneor more glycans to the surface glycoproteins. For example, glycosylation(e.g., chemical and/or enzymatic glycosylation) can be used to add oneor more glycans to the surface glycoproteins. In some embodiments, anextracellular vesicle having one or more modified glycoproteinsdescribed herein can be made by stabilizing one or more glycans alreadypresent on the surface glycoproteins.

An extracellular vesicle having one or more modified glycoproteinsdescribed herein can be made using any appropriate method. In someembodiments, an extracellular vesicle having one or more modifiedglycoproteins described herein can be made by removing certain glycansfrom the one or more surface glycoproteins and/or by removing theextrasomal surface portion of a surface glycoprotein as taught herein.

An extracellular vesicle described herein can be used for extracellularvesicle-mediated delivery of one or more cargos to a mammal (e.g., ahuman). In some embodiments, the extracellular vesicle used for deliveryof cargo comprises modification of one or more glycan(s) present on theouter surface of the extracellular vesicle. In some embodiments, one ormore glycan(s) present on the outer surface of the extracellular vesicleis modified by alteration, substitution, addition, and/or deletion ofone or more amino acid residues. In some embodiments, the modificationof the one or more glycan(s) is used to target the extracellular vesicleto a specific cell type. In some embodiments, the modification of theone or more glycan(s) is used to reduce and/or eliminate degradation ofthe extracellular vesicle. In some embodiments, the modification of theone or more glycan(s) is used to reduce and/or eliminate degradation ofthe extracellular vesicle via macrophages. In some embodiments, the oneor more glycan(s) is modified by alteration, substitution, and/ordeletion of one or more polypeptide sequences adjacent to arginineand/or lysine residues in a glycan(s) present on the outer surface ofthe extracellular vesicle. In some embodiments, the modification of theone or more glycan(s) is used to decrease the uptake of the exosome intointestinal cells. In some embodiments, the one or more glycan(s) ismodified by removal of galactosidase residues in a glycan(s) present onthe outer surface of an extracellular vesicle.

In some embodiments, the extracellular vesicle used for delivery ofcargo having one or more modified glycoproteins described herein can bemade by removing certain glycans from the one or more surfaceglycoproteins and/or by removing the extrasomal surface portion of asurface glycoprotein. In some embodiments, the extracellular vesiclehaving one or more modified glycoproteins is treated with a protease(e.g., Glu-C (targeting glu), trypsin (targeting arg and lys), and/orArg-C (targeting arg and lys)) to remove the extrasomal surface portionof a surface glycoprotein. In some embodiments, an extracellular vesiclehaving one or more modified glycoproteins described herein is made byintroducing one or more glycans to the surface glycoproteins. Forexample, glycosylation (e.g., chemical and/or enzymatic glycosylation)can be used to add one or more glycans to the surface glycoproteins. Insome embodiments, an extracellular vesicle having one or more modifiedglycoproteins described herein can be made by stabilizing one or moreglycans already present on the surface glycoproteins. In any of theseembodiments for delivery of one or more cargos to a mammal (e.g., ahuman), the extracellular vesicle can be an exosome, e.g., a milkexosome. In some embodiments, the extracellular vesicle is a bovine milkexosome. In some embodiments, the extracellular vesicle is a goat milkexosome. In some embodiments, the extracellular vesicle is a pig milkexosome.

In any of the embodiments described herein where the biological membraneof an EV or exosome has been modified to alter one or more glycoproteins(e.g., increase, decrease, or modify) or to alter one or more glycans(e.g., increase, decrease, or modify) on a glycoprotein in thebiological membrane of an EV or exosome, the EV or exosome having themodified biological membrane can be used to alter the delivery of themodified EV or exosome, and its corresponding cargo, to a mammalian cellor tissue. For example, the EV or exosome having modified biologicalmembrane can be used to increase or decrease the delivery, increase ordecrease the transport, increase or decrease the uptake of the EV orexosome and/or its corresponding cargo to mammalian cells and tissues.Also, the EV or exosome having modified biological membrane can be usedto selectively target a specific mammalian cell or tissue by alteringthe glycoprotein and/or glycan in its biological membrane, thusproviding targeted delivery, transport, and/or uptake of the UV orexosome and/or its cargo. Using the compositions and methods describedherein, the biological membrane of the EV or exosome can be customizedby altering the amount and content of the glycoprotein or glycan in thebiological membrane to effect any of the above-mentioned functions.

Extracellular vesicles vary in size and a given sample of vesicles willhave an average diameter with individual vesicles varying within arange. Extracellular vesicles, e.g. milk exosomes, of a particulardiameter or average diameter may be selected for use in accordance withthe present disclosure. In some embodiments, an exosome is about 20 nmto about 200 nm in diameter. In some embodiments, an exosome is about 30nm to about 190 nm or about 25 nm to about 180 nm in diameter. In someembodiments, an exosome is about 30 nm to about 170 nm in diameter. Insome embodiments, an exosome is about 40 nm to about 160 nm in diameter.In some embodiments, an exosome is about 50 nm to about 150 or about 60to about 140 nm, about 100 to about 200, about 80 to about 250, about 70to about 130, about 80 to about 120, or about 90 to about 110 nm indiameter. In some embodiments, an exosome is about 20, 25, 30, 35, 50,75, 100, 110, 125, or 150 nm in diameter. In some embodiments, anaverage exosome diameter in an exosomal composition or plurality ofexosomes isolated or derived from milk is about 20, about 25, about 30,about 35, about 50, about 75, about 100, about 110, about 125, or about150 nm; or about 20 to about 200, about 25 to about 250, about 100 toabout 200, about 80 to about 250, about 30 to about 180, about 40 toabout 170, about 50 to about 160, about 50 nm to about 150, about 60 toabout 140 nm, about 70 to about 130, about 80 to about 120, or about 90to about 110 nm in average diameter.

Cargos

An extracellular vesicle (e.g., an exosome) described herein can carryany appropriate cargo for delivery to a mammal (e.g., a human). Thecargo can be conjugated to an extracellular vesicle, embedded within anextracellular vesicle, encapsulated within an extracellular vesicle, orotherwise carried by an extracellular vesicle, or any combinationthereof. Thus, as used herein, a reference to a cargo being “present” inan extracellular vesicle or its lumen is understood to include any ofthe foregoing means of carrying the cargo.

In some embodiments, an exosome is loaded with 2-5 molecules or copiesof a single cargo or two (or more) different cargos. In someembodiments, an exosome or pharmaceutical composition thereof is loadedwith 1-4,000, 10-4,000, 50-3,500, 100-3,000, 200-2,500, 300-1,500,500-1,200, 750-1,000, 1-2,000, 1-1,000, 1-500, 10-400, 50-300, 1-250,1-100, 2-50, 2-25, 2-15, 2-10, 3-50, 3-25, 3-25, 3-10, 4-50, 4-25, 4-15,4-10, 5-50, 5-25, 5-15, or 5-10 molecules or copies of a single cargo ortwo (or more) different cargos. The cargo is endogenous or exogenous,and where two or more cargos are present each cargo is independentlyendogenous or exogenous.

Examples of endogenous (naturally-occurring) cargos include proteins andother agents found naturally in microvesicles such as milk exosomesinclude CD63, Transferrin receptor, sialic acid, mucins, Tsg101 (Tumorsusceptibility gene 101), Alix, annexin II, EFla (Translation elongationfactor 1a), CD82 (Cluster of Differentiation 82), ceramide,sphingomyelin, lipid raft markers, and PRNP (PRioN Protein), and othercargos shown in FIG. 50.

A cargo can be an endogenous cargo, an exogenous cargo, or a combinationthereof. Examples of cargos that can be conjugated, embedded,encapsulated within or otherwise carried by an extracellular vesicledescribed herein include, without limitation, nucleic acid molecules(e.g., DNA, cDNA, antisense oligonucleotides, mRNA, inhibitory RNAs(e.g., antisense RNAs, miRNAs, small interfering RNAs (siRNAs), shorthairpin RNAs (shRNAs), and agomiRs), antagomiRs, primary miRNAs(pri-miRNAs), long non-coding RNAs (lncRNAs), small nuclear RNA (snRNA),small nucleolar RNA (snoRNA), and microbial RNAs), polypeptides (e.g.,enzymes, antibodies), lipids, hormones, vitamins, minerals, smallmolecules, and pharmaceuticals, or any combination thereof. In someembodiments, the miRNA is an endogenous miRNA such as miR-29b ormiR-200c. In some embodiments, the miRNA is selected from those in Table1 below:

TABLE 1 Comparison table for top miRs Column 1 Column 2 Column 3 MinimumRPKM Minimum RPKM Types of Normal of Sonicated db_annotationbta-miR-320a|MI0012211 2363558.33 419367.6169 bta-miR-3596|MI0005453400272.8183 4673 bta-miR-423-5p|MI0005046 449665.5261 2461bta-miR-3600|MI0015943 65667.42731 305 bta-miR-186|MIMAT000381856127.64614 0 bta-miR-181a|MIMAT0003543 63842.85994 0bta-miR-148a|MIMAT0003522 37266.265 0 bta-miR-30a-5p|MIMAT000384122698.08901 160 bta-miR-378|MIMAT0009305 27048.72601 412bta-miR-200a|MIMAT0003822 18954.00743 0 bta-miR-378|MIMAT0009305_122045.49346 397 bta-miR-26c|MI0009784|MI0015949 11491.79978 0bta-miR-26c|MI0004731 11491.79978 0 bta-miR-2285t|MI0022348 22215.968840 bta-miR-21-5p|MI0004742 3432.615519 0 bta-miR-7c|MI0005454 12387.264755 bta-miR-26b|MI0004745 2345.26561 0 bta-miR-24-3p|MI000976113282.72962 0 bta-miR-181b|MIMAT0003793 8656.160873 0

An extracellular vesicle (e.g., an exosome) described herein can includeone or more cargos, wherein the cargo(s) is a therapeutic molecule.Exemplary therapeutic molecules include small molecules of molecularweight of less than about 1000, 800, 500, or 300 amu and othertherapeutic molecules described herein. Exemplary small moleculesinclude, without limitation, antibiotics, steroids, sterols, peptides,natural products, alkaloids, terpenes, and synthetic molecules.

A therapeutic molecule can be conjugated to an extracellular vesicle,embedded within an extracellular vesicle, encapsulated within anextracellular vesicle, or otherwise carried by an extracellular vesicleor any combination thereof. Examples of therapeutic agents include,without limitation, mRNAs and/or polypeptides encoded by the mRNAs(e.g., Cre recombinase, insulin, peptide hormones, and enzymes), miRNAs,siRNAs, or miRNA antagonists of therapeutic value, nutrients that may beunstable or have low bioavailability (e.g., vitamins Bi and B12,polyunsaturated fatty acids), pharmaceuticals (e.g., antibiotics (suchas puromycin, gentamycin, and neomycin), cancer drugs (such aschemotherapeutics, immunotherapies, hormone therapies, and targetedtherapies), activators of Toll-like receptors), and molecules to bedelivered to macrophages (e.g., to remove or prevent atheroscleroticplaques, or treat macrophage-related cancers), as well as any of theother therapeutic cargo molecules provided herein.

In some embodiments, the biologic therapeutic agent is a biologic. Insome embodiments, the biologic is selected from a hormone, allergen,adjuvant, antigen, immunogen, vaccine, interferon, interleukin, growthfactor, monoclonal antibody (mAb). In some embodiments, the biologic isa polypeptide, or a peptide, such as one containing ten or more aminoacids but less than 50; a protein, such as a protein containing 50 ormore amino acids but less than 300; or a protein having a mass fromabout 10 kD to about 30 kD, or about 30 kD to about 150 or to about 300kD.

In some embodiments, the biologic is insulin or another peptide hormone.

In some embodiments, the cargo is selected from treatments forAlzheimer's Disease such as Aricept® and Excelon®; treatments for HIVsuch as ritonavir; treatments for Parkinson's Disease such asL-DOPA/carbidopa, entacapone, ropinrole, pramipexole, bromocriptine,pergolide, trihexephendyl, and amantadine; agents for treating MultipleSclerosis (MS) such as beta interferon (e.g., Avonex® and Rebif®),Copaxone®, and mitoxantrone; treatments for asthma such as albuterol andSingulair®; agents for treating schizophrenia such as zyprexa,risperdal, seroquel, and haloperidol; anti-inflammatory agents such ascorticosteroids, TNF blockers, IL-1 RA, azathioprine, cyclophosphamide,and sulfasalazine; immunomodulatory and immunosuppressive agents such ascyclosporin, tacrolimus, rapamycin, mycophenolate mofetil, interferons,corticosteroids, cyclophophamide, azathioprine, and sulfasalazine;neurotrophic factors such as acetylcholinesterase inhibitors, MAOinhibitors, interferons, anti-convulsants, ion channel blockers,riluzole, and anti-Parkinsonian agents; agents for treatingcardiovascular disease such as beta-blockers, ACE inhibitors, diuretics,nitrates, calcium channel blockers, and statins; agents for treatingliver disease such as corticosteroids, cholestyramine, interferons, andanti-viral agents; agents for treating blood disorders such ascorticosteroids, anti-leukemic agents, and growth factors; agents thatprolong or improve pharmacokinetics such as cytochrome P450 inhibitors(i.e., inhibitors of metabolic breakdown) and CYP3A4 inhibitors (e.g.,ketokenozole and ritonavir), and agents for treating immunodeficiencydisorders such as gamma globulin.

In some embodiments, the cargo is a steroid or anti-inflammatory agentsuch as glucocorticosteroids such as budesonide, beclamethasonedipropionate, fluticasone propionate, ciclesonide or mometasone furoate;non-steroidal glucocorticoid receptor agonists; LTB4 antagonists suchLY293111, CGS025019C, CP-195543, SC-53228, BIIL 284, ONO 4057, SB209247; LTD4 antagonists such as montelukast and zafirlukast; PDE4inhibitors such cilomilast (Ariflo® GlaxoSmithKline), Roflumilast (BykGulden), V-11294A (Napp), BAY19-8004 (Bayer), SCH-351591(Schering-Plough), Arofylline (Almirall Prodesfarma), PD189659/PD168787(Parke-Davis), AWD-12-281 (Asta Medica), CDC-801 (Celgene),SeICID™CC-10004 (Celgene), VM554/UM565 (Vernalis), T-440 (Tanabe),KW-4490 (Kyowa Hakko Kogyo); A2a agonists; A2b antagonists; and beta-2adrenoceptor agonists such as albuterol (salbutamol), metaproterenol,terbutaline, salmeterol fenoterol, procaterol, and especially,formoterol and pharmaceutically acceptable salts thereof. Suitablebronchodilatory drugs include anticholinergic or antimuscariniccompounds, in particular ipratropium bromide, oxitropium bromide,tiotropium salts and CHF 4226 (Chiesi), and glycopyrrolate.

Suitable antihistamine drug substances include cetirizine hydrochloride,acetaminophen, clemastine fumarate, promethazine, loratidine,desloratidine, diphenhydramine and fexofenadine hydrochloride,activastine, astemizole, azelastine, ebastine, epinastine, mizolastineand tefenadine.

In some embodiments, the cargo is selected from small molecules orrecombinant biologic agents and include, for example, acetaminophen,non-steroidal anti-inflammatory drugs (NSAIDS) such as aspirin,ibuprofen, naproxen, etodolac (Lodine®) and celecoxib, colchicine(Colcrys®), corticosteroids such as prednisone, prednisolone,methylprednisolone, hydrocortisone, and the like, probenecid,allopurinol, febuxostat (Uloric®), sulfasalazine (Azulfidine®),antimalarials such as hydroxychloroquine (Plaquenil®) and chloroquine(Aralen®), methotrexate (Rheumatrex®), gold salts such as goldthioglucose (Solganal®), gold thiomalate (Myochrysine®) and auranofin(Ridaura®), D-penicillamine (Depen® or Cuprimine®), azathioprine(Imuran®), cyclophosphamide (Cytoxan®), chlorambucil (Leukeran®),cyclosporine (Sandimmune®), leflunomide (Arava®) and “anti-TNF” agentssuch as etanercept (Enbrel®), infliximab (Remicade®), golimumab(Simponi®), certolizumab pegol (Cimzia®) and adalimumab (Humira®),“anti-IL-” agents such as anakinra (Kineret®) and rilonacept(Arcalyst®), canakinumab (Ilaris®), anti-Jak inhibitors such astofacitinib, antibodies such as rituximab (Rituxan®), “anti-T-cell”agents such as abatacept (Orencia®), “anti-IL-6” agents such astocilizumab (Actemra®), diclofenac, cortisone, hyaluronic acid (Synvisc®or Hyalgan®), monoclonal antibodies such as tanezumab, anticoagulantssuch as heparin (Calcinparine® or Liquaemin®) and warfarin (Coumadin®),antidiarrheals such as diphenoxylate (Lomotil®) and loperamide(Imodium®), bile acid binding agents such as cholestyramine, alosetron(Lotronex®), lubiprostone (Amitiza®), laxatives such as Milk ofMagnesia, polyethylene glycol (MiraLax®), Dulcolax®, Correctol® andSenokot®, anticholinergics or antispasmodics such as dicyclomine(Bentyl®), Singulair®, beta-2 agonists such as albuterol (Ventolin® HFA,Proventil® HFA), levalbuterol (Xopenex®), metaproterenol (Alupent®),pirbuterol acetate (Maxair®), terbutaline sulfate (Brethaire®),salmeterol xinafoate (Serevent®) and formoterol (Foradil®),anticholinergic agents such as ipratropium bromide (Atrovent®) andtiotropium (Spiriva®), inhaled corticosteroids such as beclomethasonedipropionate (Beclovent®, Qvar®, and Vanceril®), triamcinolone acetonide(Azmacort®), mometasone (Asthmanex®), budesonide (Pulmocort®), andflunisolide (Aerobid®), Afviar®, Symbicort®, Dulera®, cromolyn sodium(Intal®), methylxanthines such as theophylline (Theo-Dur®, Theolair®,Slo-bid®, Uniphyl®, Theo-24®) and aminophylline, IgE antibodies such asomalizumab (Xolair®), nucleoside reverse transcriptase inhibitors suchas zidovudine (Retrovir®), abacavir (Ziagen®), abacavir/lamivudine(Epzicom®), abacavir/lamivudine/zidovudine (Trizivir®), didanosine(Videx®), emtricitabine (Emtriva®), lamivudine (Epivir®),lamivudine/zidovudine (Combivir®), stavudine (Zerit®), and zalcitabine(Hivid®), non-nucleoside reverse transcriptase inhibitors such asdelavirdine (Rescriptor®), efavirenz (Sustiva®), nevairapine (Viramune®)and etravirine (Intelence®), nucleotide reverse transcriptase inhibitorssuch as tenofovir (Viread®), protease inhibitors such as amprenavir(Agenerase®), atazanavir (Reyataz®), darunavir (Prezista®),fosamprenavir (Lexiva®), indinavir (Crixivan®), lopinavir and ritonavir(Kaletra®), nelfinavir (Viracept®), ritonavir (Norvir®), saquinavir(Fortovase® or Invirase®), and tipranavir (Aptivus®), entry inhibitorssuch as enfuvirtide (Fuzeon®) and maraviroc (Selzentry®), integraseinhibitors such as raltegravir (Isentress®), doxorubicin(Hydrodaunorubicin®), vincristine (Oncovin®), bortezomib (Velcade®), anddexamethasone (Decadron®) in combination with lenalidomide (Revlimid®),or any combination(s) thereof.

In some embodiments, the cargo is selected from corticosteroids such asprednisone, prednisolone, methylprednisolone, hydrocortisone, and thelike, sulfasalazine (Azulfidine®), antimalarials such ashydroxychloroquine (Plaquenil®) and chloroquine (Aralen®), methotrexate(Rheumatrex®), gold salts such as gold thioglucose (Solganal®), goldthiomalate (Myochrysine®) and auranofin (Ridaura®), D-penicillamine(Depen® or Cuprimine®), azathioprine (Imuran®), cyclophosphamide(Cytoxan®), chlorambucil (Leukeran®), cyclosporine (Sandimmune®),leflunomide (Arava®) and “anti-TNF” agents such as etanercept (Enbrel®),infliximab (Remicade®), golimumab (Simponi®), certolizumab pegol(Cimzia®) and adalimumab (Humira®), “anti-IL-1” agents such as anakinra(Kineret®) and rilonacept (Arcalyst®), antibodies such as rituximab(Rituxan®), “anti-T-cell” agents such as abatacept (Orencia®) and“anti-IL-6” agents such as tocilizumab (Actemra®); beta-2 agonists suchas albuterol (Ventolin® HFA, Proventil® HFA), levalbuterol (Xopenex®),metaproterenol (Alupent®), pirbuterol acetate (Maxair®), terbutalinesulfate (Brethaire®), salmeterol xinafoate (Serevent®) and formoterol(Foradil®), anticholinergic agents such as ipratropium bromide(Atrovent®) and tiotropium (Spiriva®), methylxanthines such astheophylline (Theo-Dur®, Theolair®, Slo-bid®, Uniphyl®, Theo-24) andaminophylline, inhaled corticosteroids such as prednisone, prednisolone,beclomethasone dipropionate (Beclovent®, Qvar®, and Vanceril®),triamcinolone acetonide (Azmacort®), mometasone (Asthmanex®), budesonide(Pulmocort®), flunisolide (Aerobid®), Afviar®, Symbicort®, and Dulera®,nucleoside reverse transcriptase inhibitors such as zidovudine(Retrovir®), abacavir (Ziagen®), abacavir/lamivudine (Epzicom®),abacavir/lamivudine/zidovudine (Trizivir®), didanosine (Videx®),emtricitabine (Emtriva®), lamivudine (Epivir®), lamivudine/zidovudine(Combivir®), stavudine (Zerit®), and zalcitabine (Hivid®),non-nucleoside reverse transcriptase inhibitors such as delavirdine(Rescriptor®), efavirenz (Sustiva®), nevairapine (Viramune®) andetravirine (Intelence®), nucleotide reverse transcriptase inhibitorssuch as tenofovir (Viread®), protease inhibitors such as amprenavir(Agenerase®), atazanavir (Reyataz®), darunavir (Prezista®),fosamprenavir (Lexiva®), indinavir (Crixivan®), lopinavir and ritonavir(Kaletra®), nelfinavir (Viracept®), ritonavir (Norvir®), saquinavir(Fortovase® or Invirase®), and tipranavir (Aptivus®), entry inhibitorssuch as enfuvirtide (Fuzeon®) and maraviroc (Selzentry®), integraseinhibitors such as raltegravir (Isentress®), rituximab (Rituxan®),cyclophosphamide (Cytoxan®), doxorubicin (Hydrodaunorubicin®),vincristine (Oncovin®), prednisone, a hedgehog signaling inhibitor, aBcl-2 inhibitor, a BTK inhibitor, a JAK/pan-JAK inhibitor, a TYK2inhibitor, a PI3K inhibitor, a SYK inhibitor, bortezomib (Velcade®), anddexamethasone (Decadron®), a hedgehog signaling inhibitor, a Bcl-2inhibitor, a BTK inhibitor, a JAK/pan-JAK inhibitor, a TYK2 inhibitor, aPI3K inhibitor, a SYK inhibitor, or lenalidomide (Revlimid®).

An extracellular vesicle (e.g., an exosome) described herein can includeone or more nutritional agents. A nutritional agent can be conjugated toan extracellular vesicle, embedded within an extracellular vesicle,encapsulated within an extracellular vesicle, or otherwise carried by anextracellular vesicle, or any combination thereof. Examples ofnutritional agents include, without limitation, vitamins, minerals,lipids, fatty acids (e.g., omega-3 fatty acids such as docosahexaenoicacid (DHA), and omega-6 fatty acids), and mRNAs and/or polypeptidesencoded by the mRNAs (e.g., insulin and enzymes).

An extracellular vesicle (e.g., an exosome) described herein also caninclude one or more detectable labels. A detectable label can beconjugated to an extracellular vesicle, embedded within an extracellularvesicle, encapsulated within an extracellular vesicle, or otherwisecarried by an extracellular vesicle or any combination thereof. Examplesof detectable molecules include, without limitation, bioluminescentlabel (e.g., luciferase), fluorescent molecules (e.g., GFP and mCherry),radionuclide molecules, biotin, and surface antigens. In someembodiments, an mRNA expressing a detectable label is encapsulatedwithin an extracellular vesicle such that delivery (e.g., temporaland/or spatial delivery) of the encapsulated RNA can be monitored in amammal.

In embodiments where an extracellular vesicle described herein containsan exogenous cargo, the exogenous cargo can be loaded (e.g., conjugatedto an extracellular vesicle, embedded within an extracellular vesicle,encapsulated within an extracellular vesicle, or otherwise carried by anextracellular vesicles, or any combination thereof) with the exogenouscargo using any appropriate method. In some embodiments, anextracellular vesicle described herein can be loaded with an exogenouscargo by, for example, mixing cargos and exosomes, electroporation,calcium precipitation, amphipathic molecules, or alcohol-based solvents.

In some embodiments, an extracellular vesicle (e.g., an exosome)conjugated to, embedding, encapsulating, or otherwise carrying one ormore cargos described herein can be administered to a mammal (e.g., ahuman). Methods of administration include, for example, subcutaneous,intraperitoneal, intravenous, and oral administration.

Methods of Using

This disclosure also provides methods of using an extracellular vesicle(e.g., an exosome) comprising one or more cargos described herein. Insome embodiments, an exosome described herein can be administered to amammal, e.g., human, to deliver one or more cargoes to a receptor cellin the mammal. The cargo of the exosome can be delivered over shortdistances (e.g., a receptor cell near the site of administration) orover long distances (e.g., a receptor cell in a distant tissue). In someembodiments, the cargo of the exosome can be delivered to a mammaliancell (e.g., a human cell) by endocytosis.

An extracellular vesicle (e.g., an exosome) comprising one or morecargos described herein can be administered to any type of mammal. Insome embodiments, an extracellular vesicle comprising one or more cargosdescribed herein can be administered to humans and other primates suchas monkeys as described herein. In some embodiments, an extracellularvesicle comprising one or more cargos described herein can beadministered to dogs, cats, horses, cows, pigs, sheep, rabbits, mice,and rats as described herein. In some embodiments wherein theextracellular vesicle is a bovine milk exosome, it can be administeredto a non-bovine mammalian species. For example, a bovine milk exosomedescribed herein can be administered to a human.

In some embodiments, an extracellular vesicle, e.g., exosome, describedherein can be administered to a mammal, e.g., human, to deliver one ormore cargoes to a receptor cell in the mammal. A receptor cell can beany appropriate receptor cell. Examples of receptor cells to which cargoof an exosome can be delivered include, without limitation, intestinalcells, endothelial cells (e.g., venous endothelial cells), immune cells(e.g., circulating immune cells), macrophages, intestinal mucosa (e.g.,small intestinal mucosa), peripheral tissue (e.g., liver, spleen, lung,brain, kidneys, pancreas) cells, diseased cells (e.g., cancer cells),and fetal cells (e.g., fetal cells in the womb).

In some embodiments, extracellular vesicle-mediated delivery of one ormore cargos to a mammal as described herein can be used to alter the gutmicrobiome of a mammal. Examples of cargos that can be used for alteringthe gut microbiome of a mammal include, without limitation, nucleicacids (e.g., RNAs), carbohydrates, lipids, and proteins. In someembodiments, administration of exosomes to a mammal can decrease therelative abundance of a bacterial species present in the gut microbiome.For example, the relative abundance of Firmicute classes Clostridia(e.g., Ruminococcaceae), and/or Verrucomicrobia classes (e.g.,Verrucomicrobiae (such as Muciniphila species)) can be decreased by theexosome-mediated delivery of one or more cargos to a mammalian cell. Insome embodiments, administration of exosomes to a mammal can increasethe relative abundance of a bacterial species present in the gutmicrobiome. For example, the relative abundance of Firmicute classes(e.g., Clostridia (such as Clostridiales species)) orerysipelotrichaceae can be increased by the exosome-mediated delivery ofone or more cargos to a mammalian cell, such as an endogenous orexogenous miRNA or mRNA. In some embodiments, the cargo is a vitamin ornutritional supplement.

In some embodiments, extracellular vesicle-mediated delivery of one ormore cargos to a mammal as described herein can be used to regulate(e.g., increase or decrease) the immune response of a mammal. Examplesof cargos that can be used to regulate the immune response of a mammalinclude, without limitation, proteins, glycoproteins, lectins, andnucleic acid molecules. In some embodiments, nucleic acid molecule(e.g., RNA) cargoes in exosomes can bind to Toll-like receptors toregulate the immune response of a mammal. Toll-like receptors (TLRs) area class of proteins that plays a key role in the innate immune system.They are single, membrane-spanning, non-catalytic receptors expressed onmacrophages and dendritic cells that recognize structurally conservedmolecules derived from microbes. Microbes that have breached physicalbarriers such as the skin or intestinal tract mucosa are recognized byTLRs, which activate immune cell responses. The TLRs include TLR1, TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, andTLR13. This, in some embodiments, the exosome comprises cargo that bindsto a TRL selected from: TRL1, TRL2, TRL3, TRL4, TRL5, TRL6, TLR7, TLR8,TLR9, TLR10, TLR11, TLR12, TLR13, and combinations thereof. In someembodiments, administration of bovine milk exosomes comprising cargo toa mammal can increase an immune response in a mammal. For example, themammal's anti-viral and/or pro-inflammatory response can be increased bythe exosome-mediated delivery of one or more cargos to a mammalian cell.

In some embodiments, extracellular vesicle-mediated delivery of one ormore cargos to a mammal as described herein can be used to enhance thefertility or fecundity of a mammal. Examples of cargos that can be usedfor enhancing the fertility of a mammal include, without limitation,endogenous or exogenous miRNAs or mRNAs.

In some embodiments, extracellular vesicle-mediated delivery of one ormore cargos to a mammal as described herein can be used to alter (e.g.,increase or decrease) the metabolism of a mammalian cell. Examples ofcargos that can be used for altering the metabolism of a mammalian cellinclude, without limitation, nucleic acid molecules, lipids, andpolypeptides. In some embodiments, administration of an exosome, e.g.,bovine milk exosome to a mammal can decrease purine metabolism in amammalian cell. In some embodiments, administration of an exosome, e.g.bovine milk exosome to a mammal can decrease amino acid metabolism in amammalian cell. For example, the metabolism of leucine, phenylalanine,alanine, lysine (e.g., leucyl-lysine), and/or isoleucine (e.g.,gamma-glutamyl-isoleucine) can be altered by the exosome-mediateddelivery of one or more cargos to a mammalian cell. In some embodiments,the cargo is an endogenous or exogenous miRNA or mRNA.

In some embodiments, extracellular vesicle-mediated delivery of one ormore cargos to a mammal as described herein can be used to alter (e.g.,increase or decrease) the gene expression of a mammalian cell. Examplesof cargos that can be used for altering the gene expression of amammalian cell include, without limitation, nucleic acid molecules,lipids, and polypeptides. In some embodiments, administration ofexosomes to a mammal can decrease branched-chain amino acid (BCAA)expression in a mammal cell. For example, the expression of BCAT1,BCAT2, and/or genes encoding enzymes involved in purine metabolism canbe altered by the exosome-mediated delivery of one or more cargos to amammalian cell.

In some embodiments, extracellular vesicle-mediated delivery of one ormore cargos to a mammal as described herein can be used to increase themuscle strength of a mammalian cell. Examples of cargos that can be usedfor increasing the muscle strength of a mammal include, withoutlimitation, nucleic acid molecules, lipids, and polypeptides.

In some embodiments, extracellular vesicle-mediated delivery of one ormore cargos to a mammal as described herein can be used to enhanceneurological processes or treat cognitive impairment of a mammal.Examples of cargos that can be used for enhancing neurological processesor treating cognitive impairment of a mammal include, withoutlimitation, nucleic acid molecules, lipids, and polypeptides. Forexample, administration of exosomes to a mammal can increasesensorimotor gating and/or cognitive performance (e.g., spatial learningand memory) in a mammal.

In some embodiments, extracellular vesicle-mediated delivery of one ormore cargos to a mammal as described herein can be used to treat amammal having a disease. Examples of cargos that can be used fortreating a mammal having a disease include, without limitation, nucleicacid molecules, lipids, and polypeptides. For example, administration ofexosomes to a mammal can be used to treat a mammal having sarcopenia,muscle loss after injury, atherosclerosis, cancer, immune diseases,impaired fecundity, and/or cognitive impairment (e.g., memory loss).

In some embodiments, the present disclosure provides a method ofaltering the uptake of a milk exosome into a mammalian cell or tissue,said exosome having a biological membrane comprising one or moreglycoprotein(s), comprising modifying the biological membrane of theexosome. In some embodiments, the uptake of the milk exosome into amammalian cell or tissue is increased. In some embodiments, the uptakeof the milk exosome into a mammalian cell or tissue is decreased. Insome embodiments, the uptake of the milk exosome into a mammalian cellor tissue is selectively increased in a targeted mammalian cell ortissue. In some embodiments, the uptake of the milk exosome into amammalian cell or tissue is selectively decreased in a targetedmammalian cell or tissue. In some embodiments, the present disclosureprovides a method of targeting a milk exosome to a selected mammaliancell or tissue, said exosome having a biological membrane comprising oneor more glycoprotein(s), comprising modifying the biological membrane ofthe exosome. Any of these methods can be effected by using any of theUVs or exosomes described herein.

In other embodiments, a method of correcting dysbiosis or improving thegut microbiome or gut health of a subject is provided, comprisingadministering to said subject an effective amount of any of the exosomesprovided herein. In other embodiments, a method of treating inflammatorybowel disease in a subject is provided, comprising administering to asubject in need thereof an effective amount of any of the exosomesdescribed herein. In other embodiments, a method of treating obesity ina subject, is provided comprising administering to a subject in needthereof an effective amount of any of the exosomes described herein, Inother embodiments, a method of treating non-alcoholic fatty liver in asubject is provided, comprising administering to a subject in needthereof an effective amount of any of the exosomes provided herein. Inother embodiments, a method of increasing muscle strength, enhancingsensorimotor gating or cognitive performance, or increasing fertility orfecundity in a subject is provided, comprising administering to saidsubject an effective amount of any of the exosome provided herein or anutritional supplement provided herein. In other embodiments, a methodof treating sarcopenia, muscle loss after injury, atherosclerosis,cancer, an immune disease, impaired fecundity, or cognitive impairmentis provided, comprising administering to a subject in need thereof anyof the exosomes provided herein or a nutritional supplement providedherein.

Humans and microbes have established a symbiotic association over time,and perturbations in this association have been linked to severalimmune-mediated inflammatory diseases (IMID) including inflammatorybowel disease, rheumatoid arthritis, and multiple sclerosis. IMID is aterm used to describe a group of chronic, highly disabling diseases thataffect different organ systems. Though a cornerstone commonality betweenIMID is the idiopathic nature of disease, a considerable portion oftheir pathobiology overlaps including epidemiological co-occurrence,genetic susceptibility loci and environmental risk factors. At present,it is clear that persons with an IMID are at an increased risk fordeveloping comorbidities, including additional IMID. Advancements insequencing technologies and a parallel explosion of 16S rDNA andmetagenomics community profiling studies have allowed for thecharacterization of microbiomes throughout the human body including thegut, in a myriad of human diseases and in health. See, e.g., Forbes, J.D. et al., Front Microbiol. 2016, 7, 1081.

Most IMID are highly prevalent in well-developed industrializedcountries; in Western populations the prevalence of IMID isapproximately 5-8% and encompasses over 100 different clinical diseasesincluding inflammatory bowel disease (IBD), multiple sclerosis (MS),rheumatoid arthritis (RA), ankylosing spondylitis (AS), systemic lupuserythematosus (SLE), and psoriasis/psoriatic arthritis. The healthyhuman gut is dominated by the presence of four bacterial phyla:Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria withBacteroidetes and Firmicutes accounting for a large majority of endemicbacteria in the gut. Within the healthy human gut, the phylum Firmicutesare divided into two major classes of Gram-positive bacteria: Bacilliand Clostridia (primarily Clostridium cluster IV and Clostridium XIVa).The Bacteroidetes are Gram-negative bacteria, of which the Bacteroidesrepresents one of the most abundant genera in the gut. Altered communitycomposition has been established in a number of gastrointestinaldiseases: IBD, celiac disease, irritable bowel syndrome, functionaldyspepsia, antibiotic-associated diarrhea, tropical enteropathy, andothers. Accumulating evidence proposes that dysbiosis of the intestinalmicrobiota is not limited to gastrointestinal diseases therebysuggesting that gut bacteria can affect the systemic immunologicalresponse. A number of studies have investigated gut dysbiosis inrelation to obesity, diabetes, chronic periodontitis, vaginosis, atopicdiseases, non-alcoholic steatohepatitis (NASH), Alzheimer's disease, andothers. Forbes, J. D. et al., Front Microbiol. 2016, 7, 1081.

For example, in IBD, studies frequently document an overall reduction ofdiversity, the total number of species in a community. In fact, datafrom the MetaHIT consortium suggest that persons with IBD harbor onaverage 25% fewer microbial genes than healthy persons. Diversity isreduced in the fecal and mucosal microbiomes of IBD and has also beendocumented among monozygotic twins discordant for Crohn's Disease (CD).Decreased diversity has been attributed to shifts in the abundance ofthe Firmicutes, and more specifically the Clostridium leptum and C.coccoides group (Manichanh et al., Gut. 2006 February; 55(2):205-11).Likewise, utilizing a custom phylogenetic microarray Kang et al. (2010)reported some bacteria belonging to the Firmicutes phylum includingEubacterium rectale of the Lachnospiraceae and Ruminococcus albus, R.callidus, R. bromii, and F. prausnitzii of the Ruminococcaceae were 5-to 10-fold more abundant in healthy persons compared to CD. Kang et al.,Inflamm Bowel Dis. 2010 December; 16(12):2034-42.

Bacteroides is the most dominant genus in Western microbiotas and canboth positively and negatively affect the host. Generally, while theoverall abundance of the order Bacteroidales is increased in IBD, incertain circumstances particular species may be reduced; Parabacteroidesdistasonis is significantly decreased in inflamed IBD mucosa (Zitomerskyet al., PLoS One. 2013; 8(6):e63686). Pathogenic bacteria including E.coli, and Shigella, and others such as Rhodococcus and Stenotrophomonasmaltophilia are increasingly observed in IBD. Other pathobionts withpotential roles in the disease course include Prevotellaceae, C.difficile, Klebsiella pneumoniae, Proteus mirabilis, and Helicobacterhepaticus.

The relative abundance of the Enterobacteriaceae in persons with IBD(Kolho et al., Am J Gastroenterol. 2015 June; 110(6):921-30) and mousemodels of IBD (Nagao-Kitamoto et al., CMGH Cell. Mol. Gastroenterol.Hepatol. 2 468-481) is increased. Much research has focused on the roleof E. coli, specifically AIEC, in IBD etiology; AIEC strains have beenisolated from ileal-involving CD tissue and the genera Escherichia andShigella (indistinguishable by 16S analysis) are highly enriched inpatients with IBD and this enrichment was more pronounced in mucosalsamples versus stool samples.

Pharmaceutical Compositions and Formulations; Routes of Delivery

Cargos may be in the form of pharmaceutically acceptable salts. As usedherein, the term “pharmaceutically acceptable salt” refers to thosesalts which are, within the scope of sound medical judgment, suitablefor use in contact with the tissues of humans and lower animals withoutundue toxicity, irritation, allergic response and the like, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid ormalonic acid or by using other methods used in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate,propionate, stearate, succinate, sulfate, tartrate, thiocyanate,p-toluenesulfonate, undecanoate, valerate salts, and the like.

Salts derived from appropriate bases include alkali metal, alkalineearth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representative alkali oralkaline earth metal salts include sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and arylsulfonate.

In some embodiments, an extracellular vesicle (e.g., an exosome)encapsulating one or more cargos described herein can be formulated as apharmaceutical composition and/or a nutritional supplement (e.g., a“nutraceutical”). For example, an extracellular vesicle preparation canbe formulated to contain a pharmaceutically and/or nutritionallyacceptable carrier for administration to a mammal. Examples of suchcarriers include, without limitation, sterile aqueous or non-aqueoussolutions, solvents, suspensions, and emulsions. Examples of non-aqueoussolvents include, without limitation, propylene glycol, polyethyleneglycol, vegetable oils, and organic esters. Aqueous carriers include,without limitation, water, alcohol, saline, and buffered solutions.Acceptable carriers also can include physiologically acceptable aqueousvehicles (e.g., physiological saline) or other carriers used for oraladministration. In some embodiments, a nutritional supplement containingexosomes as described herein can be used in infant formulas.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound (or microvesicle,as the case may be) with which it is formulated. Pharmaceuticallyacceptable carriers, adjuvants or vehicles that may be used in thecompositions of this invention include, but are not limited to, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

Compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

Pharmaceutically acceptable compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The cargo can also be in micro-encapsulated form with one or moreexcipients as noted above. The solid dosage forms of tablets, dragees,capsules, pills, and granules can be prepared with coatings and shellssuch as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the cargo may be admixed with at least one inert diluentsuch as sucrose, lactose or starch. Such dosage forms may also comprise,as is normal practice, additional substances other than inert diluents,e.g., tableting lubricants and other tableting aids such a magnesiumstearate and microcrystalline cellulose. In the case of capsules,tablets and pills, the dosage forms may also comprise buffering agents.They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Alternatively, pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

Pharmaceutically acceptable compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, provided pharmaceutically acceptablecompositions may be formulated in a suitable ointment containing theactive component suspended or dissolved in one or more carriers.Carriers for topical administration of compounds of this inventioninclude, but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Alternatively, providedpharmaceutically acceptable compositions can be formulated in a suitablelotion or cream containing the active components suspended or dissolvedin one or more pharmaceutically acceptable carriers. Suitable carriersinclude, but are not limited to, mineral oil, sorbitan monostearate,polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol,benzyl alcohol and water.

For ophthalmic use, provided pharmaceutically acceptable compositionsmay be formulated as micronized suspensions in isotonic, pH adjustedsterile saline, or, preferably, as solutions in isotonic, pH adjustedsterile saline, either with or without a preservative such asbenzylalkonium chloride. Alternatively, for ophthalmic uses, thepharmaceutically acceptable compositions may be formulated in anointment such as petrolatum.

Pharmaceutically acceptable compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

In some embodiments, pharmaceutically acceptable compositions of thisinvention are formulated for oral administration. Such formulations maybe administered with or without food. In some embodiments,pharmaceutically acceptable compositions of this invention areadministered without food. In other embodiments, pharmaceuticallyacceptable compositions of this invention are administered with food.

The amount of compounds of the present invention that may be combinedwith the carrier materials to produce a composition in a single dosageform will vary depending upon the subject treated and the particularmode of administration. Preferably, provided compositions should beformulated so that a dosage of between 0.01-100 mg/kg body weight/day ofthe inhibitor can be administered to a patient receiving thesecompositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

A pharmaceutical composition and/or a nutritional supplement can beformulated for administration in solid or liquid form including, withoutlimitation, sterile solutions, suspensions, sustained-releaseformulations, tablets, capsules, pills, powders, and granules.

Methods of administration include, for example, subcutaneous,intraperitoneal, intravenous, and oral administration.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1. Methods Used in Milk Exosome Studies

A. Isolation of Exosomes from Bovine Milk

Cow's milk (1% fat or skim milk) was obtained from a local grocerystore. Exosomes were isolated from cow's milk using ultracentrifugation.The milk was centrifuged at either 12,000×g at 4° C. for 30 min or13,200×g at 4° C. for 30 min to remove somatic cells and debris. Thesupernatant was mixed 1:1 (by vol) with 250 mM EDTA (pH 7.0) on ice for15 min to precipitate milk casein and exosomes coated with casein. Thesuspension was ultracentrifuged at either 80,000×g or 100,000×g at 4° C.for 60 min (F37L-8 3 100 rotor; Thermo Scientific) to removeprecipitated protein, milk fat globules, and microvesicles larger thanthe exosomes. The supernatant was ultracentrifuged at 120,000×g for 90min at 4° C. to collect exosomes. The exosome pellet was resuspended ina small volume of sterile PBS containing 0.01% sodium azide, filteredtwice through a 0.22-mm membrane filter (Milex), and stored at 4° C. ifused the same day or −20° C. for up to 5 days. Zempleni et al., TheIntestinal Transport of Bovine Milk Exosomes Is Mediated by Endocytosisin Human Colon Carcinoma Caco-2 Cells and Rat Small Intestinal IEC-6Cells, J Nutr 2015; 145:2201-6.

B. Confirmation of Exosome Identity and Purity

The identity, purity, and integrity of the isolated exosomes wereconfirmed using nanoparticle tracker, western blot, and transmissionelectron microscopy. Absence of aggregation and exosome purity wereassessed as recommended by the International Society for ExtracellularVesicles. Lotvall J, et al. Minimal experimental requirements fordefinition of extracellular vesicles and their functions: a positionstatement from the International Society for Extracellular Vesicles. JExtracell Vesicles 2014; 3:26913. Briefly, absence of exosomeaggregation was confirmed using transmission electron microscopy(Hitachi H7500; Hitachi) in the Microscopy Core Facility at theUniversity of Nebraska-Lincoln. ImageJ was used to analyze the exosomesize distribution, which averaged 69±20 nm in diameter.

Exosome purity and identity were confirmed using whole protein extractsfrom exosomes resolved by gel electrophoresis (10 μg protein/lane) asdescribed in: An K, et al. Exosomes neutralizesynaptic-plasticity-disrupting activity of Abeta assemblies in vivo. MolBrain 2013; 6:47. Membranes were probed using mouse anti-bovine CD63(catalog no. MCA2042GA; AbD Serotec), mouse anti-CD9 (catalog no.ab61873; Abcam), goat anti-bovine Alix (catalog no. sc-49268; Santa CruzBiotechnology) as markers for exosomes, rabbit antiserum to bovineα-s1-casein as a marker for the animal species of exosome origin, andgoat anti-bovine histone H3 (catalog no. sc-8654; Santa CruzBiotechnology) as a negative control (all at 1,000-fold dilutions).Protein extracts were run on the same gel, membranes were cut forprobing with the cited antibodies, and images were reassembled afterprobing. Bands were visualized using an Odyssey infrared imaging system(Licor) and RDye 800CW-labeled secondary antibodies (50,000-folddilution; catalog nos. 926-32210, 926-32214, and 926-32211; Licor). FIG.2A shows milk exosome preparations from cow's milk. Exosome extractswere probed using anti-CD63, anti-CD9, anti-Alix, anti-α-s1 casein, andantihistone H3. Protein extracts were run on the same gel, membraneswere cut for probing with the cited antibodies, and images werereassembled after probing. FIG. 2B shows transmission electronmicroscope images of exosome preparations. The large field image wasobtained with a 15,000-fold magnification; the insert depicts a singleparticle selected from the same image. FIGS. 3A-2D shows proteinsexpressed in exosome cytoplasmic extract and membrane protein extract ofmilk exosome. Exosome extracts were probed using GAPDH, ALIX, anti-CD63,and anti-CD9 (ALIX, CD63, and CD9 are markers for exosomes).Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic enzymeand is a marker for cytoplasmic proteins. Protein extracts were run onthe same gel, membranes were cut for probing with the cited antibodies,and images were reassembled after probing.

Anti-bovine α-s1-casein was raised in rabbits (Cocalico) using anacetylated casein-based sequence and coupled to keyhole limpethemocyanin through a C-terminal cysteine. FIG. 4 shows gelelectrophoresis and membrane blot results in which the anti-serumproduced bands of the expected size with cow's milk exosomes (lane 1),cow's milk (lane 2), and α-s1 casein peptide (lane 5), but not withnegative controls such as human breast milk (lane 4), an unrelatedsynthetic peptide (platelet glycoprotein 1) (lane 4), and exosomes fromchicken egg yolk (lane 6). Ten micrograms of milk and exosome proteinwere loaded per lane, whereas only 1 μg of synthetic peptides wereloaded. M, molecular weight markers.

C. Other Methods

For studies in cell cultures, exosomes were labeled with FM-464(Molecular Probes) as known in the field. Unbound fluorophore wasremoved by pelleting the exosomes at 120,000 g and 4° C. for 90 min,followed by three wash and ultracentrifugation cycles with sterilephosphate-buffered saline. For studies in mice, exosomes were labeledwith a cyanine-based fluorophore, 1,1′-dioctadecyltetramethylindotricarbocyanine iodide (DiR) as described elsewhere herein. Exosomeintegrity and absence of aggregation was confirmed by transmissionelectron microscopy. The concentration of exosome protein was measuredusing a Nanodrop-1000 spectrophotometer (NanoDrop Technologies), andexosomes were diluted with F-12K media to produce the desired proteinconcentration.

Example 2. Non-Sonicated Versus Sonicated Microvesicles

Sonication of microvesicles, e.g., exosomes, has an effect on exosomesize, physical properties, and functional properties of themicrovesicle. The effects of sonification on the physical/structuralproperties as well as the functional properties have been studied andare reported here. Sonication of exosomes is known in the art, see,e.g., J Nutr. 2014, October; 144(10):1495-500.

Sonication of microvesicles, e.g., exosomes, affects the physical (e.g.,structural) properties of the microvesicles. For example, FIGS. 5-8 showthat the average diameter of microvesicles, e.g. milk exosomes, aremodulated by sonication. Specifically, sonification affects thestructure of the exosomes such that the structures are more variable,resulting in a greater average diameter in sonicated exosomes ascompared with non-sonicated exosomes. FIG. 5 shows nano trackinganalyzer size analysis of milk exosomes indicating an exosome size rangeunder 200 nm. FIG. 6 shows nano tracking analyzer size analysis ofsonicated milk exosomes indicating an exosome size range of up to 300nm. Further structural studies showed that natural (non-sonicated)exosomes have smooth, well-formed surfaces whereas sonicated exosomeshave rough, more irregular surfaces and some clustering or aggregation(FIGS. 7A and 8A showing the atomic force microscopy results ofnon-sonicated and sonicated exosomes, respectively). FIG. 7B shows thatnatural (non-sonicated) exosomes have a diameter range of 120-200 nm.FIG. 8B shows that sonicated exosomes have a diameter range of 120-250nm. In addition, sonification can affect the membrane properties of theexosome, including, for example, altering the types and amount ofglycoproteins found in/on the membrane or surface of exosomes and/or thetypes, extent, and/or amount of glycosylation of the surfaceglycoproteins.

Sonication also affects the functional properties of the microvesicle,e.g., exosomes, for example, by altering its delivery and transportproperties, altering its cellular targeting, altering its cellularuptake, altering its stability against degradation, altering itsdelivery of endogenous cargo, altering its delivery of exogenous cargo,as well as other functional biological properties. In some instances,sonication modifies the microvesicle surface glycoprotein, which may inturn modify the cellular uptake of the microvesicle. In some instances,sonication reduces the number of surface glycoproteins and/or surfaceglycans presented on the outer surface of the microvesicle. In someinstances, sonication increases microvesicle stability againstdegradation, such as by macrophages. In some instances, sonicationdecreases the rate of cellular uptake of the microvesicle. For example,FIGS. 9A, 9B, and FIG. 10 show that sonication of milk exosomesdecreases the uptake of exosomes into mammalian cells, e.g., Caco-2cells (FIG. 9A), FH cells (FIG. 9B), and U937 cells (FIG. 10). In someinstances, sonication affects the rate of delivery of the microvesiclecargo, e.g., endogenous and/or exosome cargo.

Sonication may also be used to empty endogenous microvesicle contents.Sonication may also be used to load an exogenous cargo. See, e.g., Luan,X. et al., “Engineering exosomes as refined biological nanoplatforms fordrug delivery,” Acta Pharmacologica Sinica 2017, 38: 754-763, herebyincorporated by reference.

Example 3. Distribution of Bovine Milk Exosomes in Mammalian Tissues

Until recently, miRNAs have been considered endogenous regulators ofgenes, i.e., miRNAs synthesized in a given organism regulate theexpression of genes in that host. However, studies we have performedrefute this paradigm and provide strong evidence that 1) humans absorbbiologically meaningful amounts of miRNAs from nutritionally relevantdoses of cow's milk, 2) milk miRNAs are delivered to peripheral humantissues, 3) physiological concentrations of milk miRNAs affect humangene expression in vivo and in cell cultures, and 4) endogenoussynthesis of miRNAs does not compensate for dietary miRNAs deficiency inmice. These discoveries were corroborated in a recent report byinvestigators from the National Institutes of Health (NIH)-supportedGenboree database, who detected numerous dietary miRNAs in 6.8 billionsequencing reads from 528 human samples. Our studies further detectedbovine-specific miRNAs in human plasma following milk consumption usingnext generation sequencing (“Computational Characterization of ExogenousMicroRNAs that Can Be Transferred into Human Circulation,” Shu J, ChiangK, Zempleni J, Cui J. PLoS One. 2015 Nov. 3; 10(11):e0140587.). Otherstudies suggest that human and rat intestinal cells transport cow's milkexosomes by endocytosis and that milk exosomes may cross the intestinalmucosa without repackaging in mice and enter the peripheral circulationin intact form. On the basis of these previous observations it isreasonable to propose that a fraction of dietary exosomes from foods ofanimal origin enters the peripheral circulation, and that mechanismsexist for the transfer of these exosomes across vascular endothelia.Further tests were performed to demonstrate that human vascularendothelial cells transport cow's milk exosomes by a carrier mediatedprocess similar to the mechanism reported for the uptake of exosomes inintestinal cells, i.e., endocytosis.

A. Human Vascular Endothelial Cells Transport Foreign Exosomes fromCow's Milk by Endocytosis

The transport of milk exosomes was studied to determine the mechanism bywhich dietary exosomes and their cargo are delivered to peripheraltissues. Using human umbilical vein endothelial cells and fluorophorelabeled exosomes isolated from cow's milk studies were performed todemonstrate that human vascular endothelial cells transport milkexosomes via endocytosis. Exosome uptake followed Michaelis-Mentenkinetics (V_(max)=0.057±0.004 ng exosome protein×40,000 cells/h;K_(m)=17.97±3.84 μg exosomal protein/200 μl media) and decreased by 80%when the incubation temperature was lowered from 37° C. to 4° C. Whenexosome surface proteins were removed by treatment with proteinase K, ortransport was measured in the presence of the carbohydrate competitorD-galactose or measured in the presence of excess unlabeled exosomes,transport rates decreased by 45% to 80% compared with controls.Treatment with an inhibitor of endocytosis, cytochalasin D, caused a 50%decrease in transport. When fluorophore-labeled exosomes wereadministered retro-orbitally, exosomes accumulated in liver, spleen, andlungs in mice. These studies are described in greater detail below anddemonstrate that human vascular endothelial cells transport bovineexosomes by endocytosis which is an important step in the delivery ofdietary exosomes and their cargo to peripheral tissues. Further, exosomesurface proteins and their carbohydrate content play a significant rolein milk exosome transport.

(i) Cell Culture

Exosomes were isolated as described in Example 1. Human umbilical veinendothelial cells (HUVEC, passages 38-45) were purchased from AmericanType Culture Collection (CRL-1730) and cultured in F-12K medium,supplemented with 0.04 mg/ml endothelial cell growth supplement, 0.1mg/ml heparin, 100,000 U/1 penicillin and 100 mg/l streptomycin (allfrom Sigma), and 10% exosome-free fetal bovine serum in a humidifiedatmosphere at 5% CO₂ and 37° C. Exosome-free fetal bovine serum wasprepared by sonicating the serum in a water bath for 1 h to disruptmembranes, which granted milk RNases access to exosome RNAs and causedthe degradation of 60%, 65%, and 86% of miR-15b, miR-21, and miR-200c,respectively. miRNAs were quantified in exosome extracts by usingquantitative real-time PCR, U6 snRNA for normalization, and miSPIKE asinternal standard (IDT Technologies), and miRNA-specific PCR primers asdescribed previously. Media were replaced with fresh media every 48 h.

(ii) Protocols

In a typical experiment, 15×10³ HUVECs were seeded per well in a 96-wellplate and allowed to adhere overnight. Fluorophore-labeled exosomes wereadded to the wells to produce the desired concentration of exosomeprotein. Cells were incubated for various lengths of time. Media wereremoved and cells were washed three times with sterile PBS to removeextracellular exosomes. Controls were prepared by washing the cellsimmediately after addition of exosomes. Cell fluorescence (excitation560 nm, emission 645 nm) was measured in a microplate fluorescencedetector (BioTek). Cells were harvested using trypsin and counted usinga hemocytometer. Units of fluorescence were converted into mass ofexosome bound by labeling a known mass of exosomes (protein) withfluorophore, and quantifying the fluorescence after removing unboundfluorophore. In select experiments, exosome binding was determined underthe following conditions: 1) cells were treated with 5 or 10 μg/ml ofthe endocytosis inhibitor cytochalasin D (GIBCO) for 30 min beforeadding exosomes; 2) cells were treated with 150 mM carbohydratecompetitors D-glucose or D-galactose for 30 min before adding exosomes;and 3) milk exosomes were treated with 100 μg/ml of proteinase K at 37°C. for 30 min to remove surface proteins. All assays were performed inthree independent experiments, each in triplicate analyses. Bindingkinetics were modeled using the Michaelis-Menten equation and nonlinearregression (GraphPad Prism 6.0; GraphPad Software, La Jolla, Calif.).

(iii) Confirmation that Exosomes Enter the Intracellular Space

(a) Methods

The possibility that adherence to cells, rather than uptake into cells,accounted for cell fluorescence was dismissed based on the followingcell death-based and enhanced green fluorescent protein (eGFP)protocols. Milk exosomes were suspended in BTXpress electroporationbuffer (BTX, Holliston, Mass.; final concentration 100 μg/μl protein),containing zero (negative control), 56 μM, or 223 μM puromycin in a 4-mmelectroporation cuvette. Exosomes were loaded with puromycin or with amammalian eGFP expression plasmid by electroporation in a Gene PulserXcell electroporator (Bio-Rad) using 250 V, 950 μF, and infiniteresistance. Extra-exosomal puromycin was removed by ultracentrifugationand washing, and exosomes were resuspended in 300 μl of exosome-depletedcell culture media. In cell death assays, 100 μl of the suspension wereadded per well in a 96-well cell culture plate (containing ˜200 μl ofmedia) and cells were cultured for 24 h, when viability was assessedusing the MTT assay. Positive controls were created by adding puromycindirectly to the media without encapsulation in exosomes, therebyproducing a concentration of 1,837 μM puromycin. In eGFP assays, cellswere cultured in media in which eGFP-loaded milk exosomes weresubstituted for exosomes in fetal bovine serum. Expression of eGFP wasassessed by using confocal microscopy 3 days after initiation ofcultures.

(b) Results

Treatment of cells with puromycin-loaded exosomes provided compellingevidence that exosomes truly entered the intracellular space, as opposedto exosomes adhering to the cell surface. When cells were treated withpuromycin-loaded exosomes, viability decreased 75% to 84% of controls(FIG. 11A). Not surprisingly, when 1,837 μM puromycin was added directlyto media, viability decreased to 37% of puromycin-free controls(positive control). Cell death was caused by exosome-mediated deliveryof puromycin as opposed to release of puromycin from exosomes, based onthe following observation. When cells were cultured in media containingexosomes loaded with a plasmid coding for eGFP, they expressed eGFPprotein (FIG. 11B).

(iv) The uptake of milk exosomes into HUVECs is a carrier mediatedprocess.

It was established that exosome uptake was linear with time for up to 2h if 20 μg exosome protein was added to 200 μl media, i.e.,concentrations below transporter saturation (see below). Temporalpatterns were similar when 70 μg exosome protein/200 μl media were used(data not shown). Subsequent transport studies were carried out using anincubation time of 1 h. Exosome uptake followed Michaelis-Mentenkinetics: Vmax=0.057±0.004 ng exosome protein×40,000 cells/h andK_(m)=18.0±3.8 μg exosome protein/200 μl media. Exosome uptake dependedon the incubation temperature. When 100 μg of unlabeled exosomes wasadded to the cell cultures (equaling 5 times K_(m)), the uptake offluorophore-conjugated exosomes decreased to 16.8±7.2% of controls(P<0.05, n=3 biological replicates each measured in triplicate). Whencells were treated with 5 μg/ml or 10 μg/ml cytochalasin D, exosomeuptake decreased to 63.5±21.3% and 40.8±22.0%, respectively, ofcontrols, consistent with endocytosis (P<0.05, n=3).

B. Transport and Distribution of Milk Exosome into Mammalian Tissues (InVivo Studies)

(i) Protocal

To determine whether intravenously administered, DiR-labeled milkexosomes cross vascular endothelia cells and accumulate in tissues,1×10¹¹ DiR-labeled exosomes/g body wt were injected retro-orbitally(intravenously) in female C57BL/6 mice. The mice were 11 wk of age (˜25g body wt) and were fed Teklad Global 16% Protein Rodent Diet (catalogno. Teklad 2016, Envigo). Controls were injected with free DiR orunlabeled exosomes. Eighteen hours after injection, the distribution ofexosomes was assessed using an iBox small animal imaging system in livemice and excised tissues. Dissected issues were flushed with cold salineto remove circulating exosomes prior to imaging. The experiments in micewere approved by the Institutional Animal Care Program at the Universityof Nebraska-Lincoln (protocol no. 963). Statistical analysis.Homogeneity of variances was confirmed using Bartlett's test.Statistical significance of differences among treatment groups wasassessed using one-way ANOVA and Tukey-Kramer's or Dunnett's post hoctest. Analyses were performed using GraphPad Prism. Differences wereconsidered significant if P<0.05. Means±SD are reported.

Exosome purification protocol yielded preparations of nonaggregatedextracellular vesicles that were primarily composed of exosomes. Whenprotein extracts were probed with anti-CD63, anti-Alix, anti-CD9, oranti-bovine α-s1-casein, strong bands were observed in western blots; incontrast, when protein extracts were probed with anti-histone H3(negative control), no band was visible. The particle suspension waslargely free of aggregates, and the shape and contour of exosomessuggested vesicle integrity. The average particle size was 69±19.5 nm indiameter, as expected for exosomes. A few particles were detected thathad a diameter less than that of exosomes; these particles probablyrepresent small fat globules.

(ii) Results

Imaging studies in mice demonstrated that milk exosomes can crossvascular endothelial cells for delivery to tissues. Eighteen hours afterintra-orbital injection of DiR-labeled milk exosomes, the majority ofexosomes was cleared from circulation and accumulated in a region nearthe liver; no signal was detected in mice injected with free DiR orunlabeled exosomes. In excised tissues, strong signals were detectablein liver and spleen when the exposure time was 20 s, whereas traces weredetectable in intestine, stomach, and lungs only when the exposure timewas increased from 20 s to 30 s under the experimental conditions. Thesignal in liver and spleen was fully saturated at 20 s, i.e., theextension of the exposure time caused an artificial bias towards astronger signal in intestine, stomach, and lungs. FIGS. 12A-C show thedistribution of milk exosomes in C57BL/6 mice who received retro-orbitalinjections of 1,1′-dioctadecyltetramethyl indotricarbocyanine iodide(DiR)-labeled exosomes, free DiR, or unlabeled exosomes. FIG. 12A: wholemice, 18 h after injection. FIG. 12B: excised tissues, 20 s exposuretime. FIG. 12C: same sample as shown in (B) but exposure time increasedto 30 s. Scale bars depict fluorescence intensity in units of percentsaturation.

Further studies using super resolution microscopy shows that exosomesare present in the cytosol of cells following uptake by cells. In thesestudies, the nuclei of the cell was stained with Hoechst-blue, cytosol(f-Actin) with CF-594-red, and milk exosomes with PKH-67-green. FIG. 13Ashows split image for only cells, only exosomes (top left and rightpanels, respectively) and only nucleus and both exosomes and cells(bottom left and right panels, respectively). FIG. 13B shows anorthogonal cross sectional image showing the presence of exosome in thecytosol after its uptake by the cells.

Example 4: Bovine Milk Exosomes and their Corresponding Cargo arePresent in Mammalian Tissues A. Introduction

Until recently, miRNAs have been considered endogenous regulators ofgenes, i.e., miRNAs synthesized in a given organism regulate theexpression of genes in that host. However, studies we have performedrefute this paradigm and provide strong evidence that 1) humans absorbbiologically meaningful amounts of miRNAs from nutritionally relevantdoses of cow's milk, 2) milk miRNAs are delivered to peripheral humantissues, 3) physiological concentrations of milk miRNAs affect humangene expression in vivo and in cell cultures, and 4) endogenoussynthesis of miRNAs does not compensate for dietary miRNAs deficiency inmice. These discoveries were corroborated in a recent report byinvestigators from the National Institutes of Health (NIH)-supportedGenboree database, who detected numerous dietary miRNAs in 6.8 billionsequencing reads from 528 human samples. Our studies further detectedbovine-specific miRNAs in human plasma following milk consumption usingnext generation sequencing. Other studies suggest that human and ratintestinal cells transport cow's milk exosomes by endocytosis and thatmilk exosomes may cross the intestinal mucosa without repackaging inmice and enter the peripheral circulation in intact form. On the basisof these observations it is reasonable to propose that a fraction ofdietary exosomes from foods of animal origin enters the peripheralcirculation, and that mechanisms exist for the transfer of theseexosomes across vascular endothelia.

The studies in Example 3 showed that human vascular endothelial cellstransport cow's milk exosomes by a carrier mediated process similar tothe mechanism reported for the uptake of exosomes in intestinal cells,i.e., endocytosis. Specifically, the described studies showed that thetransport of cow's milk exosomes across vascular endothelial cells ismediated by endocytosis and that proteins on the surface of milkexosomes are compatible with proteins on the surface of human vascularendothelial cells. The studies and results shown in FIG. 21 furtherconfirmed that exosomes are transported into mammalian tissue followingcellular uptake.

This further corroborates the notion that dietary miRNAs have biologicalactivity in humans which has far-reaching implications for humannutrition and health. The National Cancer Institute defines bioactivecompounds as “a type of chemical found in small amounts in plants andcertain foods [. . . ]. Bioactive compounds have actions in the bodythat may promote good health. They are being studied in the preventionof [ . . . ] diseases”. Milk miRNAs meet that definition, based on ourprevious studies which suggest that cow's milk microRNAs regulate genesin circulating cells and peripheral human tissues. The studies describedherein indicate that dietary exosomes are cleared primarily by uptakeinto liver and spleen.

B. The Bioavailability and Distribution of Bovine Milk Exosomes isDistinct from that of their RNA Cargos in Mice (i) Methods

Exosomes were isolated from bovine milk using ultracentrifugation.Exosome membranes were labeled using the fluorophore, DiR, as describedelsewhere herein. RNA cargos were labeled using EXO-Glow Red in separateexperiments. Identity, integrity, fine dispersion, and count of exosomeswere assessed using transmission electron microscopy, western blots, andnanoparticle tracker (not shown). Exosomes were administered orally bygavage (1×10¹² exosomes/g) in BALB/c mice. Absorption and distributionof exosomes and their cargos was monitored at timed intervals for up to24 h using an iBox® Small Animal Imaging system and LiCor Odyssey CLx.At timed intervals, mice were euthanized and various tissues wereexcised and collected for densitometry analysis using VisionWorks®LS andImage Studio software.

(ii) Results Show Differential Distribution of Exosomes and RNA Cargo inMammalian Tissue

When DiR-labeled cow's milk exosomes were administered orally to Balb/cmice, the majority of exosomes localized in mucosa cells in the uppersmall intestine, but exosomes were also detectable in liver and spleen.Bioavailability and distribution largely depended on glycoproteins onthe surface of exosomes and intestinal cells, and on uptake bymacrophages. At t=12 h after administration of DiR-labeled exosomes bygavage, only a faint DiR signal was detected in peripheral tissues (FIG.14A). In contrast, labeled RNA was detected in all peripheral tissueswith a preference for kidneys, brain, lungs and liver (FIG. 14B: 12hours; FIG. 14C: 24 hours). FIG. 15 depicts the densitometry analysis oflabeled RNA, corrected by the signal produced by unconjugated Exo-glow.At t=24 h, the majority of absorbed exosomes accumulated in liver (FIG.16A), whereas the RNA cargo accumulated preferably in brain and kidneys(FIG. 16B).

When milk exosomes, at a concentration of 5 times the K_(m), were addedto the upper chamber in transwell plates, Caco-2 cells accumulatedmiR-29b and miR-200c in the lower chamber, and reverse transport wasminor. In transwell studies Caco-2 cells were seeded at a density of9000 cells/well with 75 mL of media in 96-well polycarbonate plates witha pore size of 0.4 mm (EMD Millipore). The cells were allowed to grow adifferentiated monolayer for 21-24 d (33). Caco-2 cell monolayerintegrity was formally confirmed using the Lucifer yellow (LY) rejectionassay according to the manufacturer's instructions (33). LY fluorescencewas measured in the transwell apical and basolateral chambers after 1 hof incubation at 37° C. In parallel experiments, Caco-2 cells werecultured in exosome-depleted media to which milk exosomes were addedback to produce a concentration of 275 mg/100 mL exosomal protein ineither the upper, apical chamber or the lower, basolateral chamber.Controls were cultured in exosome-depleted media. Aliquots of media werecollected from the upper chamber and bottom chamber after 2 h ofincubation for analysis of microRNAs. Twenty-five attomoles of internalstandard (miSPIKE Synthetic RNA; IDT Technologies) was added to samplesbefore microRNA extraction and subsequent analysis of miR-29b andmiR-200c in transwell chambers by quantitative real-time PCR andmicroRNA-specific primers; miSpike was also used for PCR calibration.Values were corrected for the internal standard to normalize forextraction efficiency.

The studies described herein show that endothelial transfer of dietarymiRNAs occurs, based on the following observations: 1) Human coloncarcinoma Caco-2 cells form tight monolayers. Our studies of intestinaltransport of milk exosomes using Caco-2 cells and transwell plates(Lucifer Yellow rejection rate 99.8%) suggest that miRNAs aretransferred.

These studies showed that exosome transport exhibited saturationkinetics at 37° C. (Michaelis constant (K_(m))=55.5 to 48.6 mg exosomalprotein/200 mL of media) and was consistent with carrier-mediatedtransport in Caco-2 cells. Exosome uptake was decreased by 61-85% underthe following conditions compared with controls in Caco-2 cells: removalof exosome and cell surface proteins by proteinase K, inhibition ofendocytosis and vesicle trafficking by synthetic inhibitors, andinhibition of glycoprotein binding by carbohydrate competitors.Transport characteristics were similar in IEC-6 cells and Caco-2 cells,except that substrate affinity and transporter capacity were lower inIEC-6 cells.

across intestinal epithelia. 2) Previous studies used miRNA reportergenes and quantified the abundance of miRNA in human kidney cellcultures, circulating primary human cells, and mouse livers todemonstrate that dietary miRNAs are delivered to cells. 3) When vesiclesare administered subcutaneously, intraperitoneally, or intravenouslythey accumulate in liver and peripheral tissues. 4) Whenfluorophore-labeled milk exosomes are administered orally (gavage) orintravenously, the majority of exosomes accumulates in liver and spleen.5) Studies using puromycin-loaded and eGFP plasmid-loaded exosomessuggest that milk exosomes enter cells, as opposed to merely adsorbingto the cell surface.

(iii) Summary

Bovine milk exosomes and their RNA cargos are bioavailable andaccumulate in distinct tissues in mice. The data suggest that bovinemilk exosomes are disassembled in the intestinal mucosa, and RNA cargosare repackaged in endogenous exosomes for transfer to tissues,preferably brain and kidneys. The data further suggest that milkexosomes are absorbed, and that a fraction of these exosomes escapesre-packaging in the intestinal mucosa and reaches tissues in intactform; the majority of exosomes accumulates in macrophages. The followingpaper demonstrated that macrophages play an important role in clearingforeign exosomes (the paper studied exosomes secreted by mouse cancercells and injected into mice): J Extracell Vesicles. 2015 Feb. 9;4:26238.

Example 5: mRNAs in Bovine Milk Exosomes are Translated into Protein

Exosomes were isolated from bovine milk as previously described herein.mRNAs were extracted, sequenced using an Illumina HiSeq 2500 platform(RNAseq) and annotated using the bovine reference genome. RabbitReticulocyte Lysates (RRL) was used to translate mRNAs usingBODIPY-labeled lysine; products were assessed using 2-D gelelectrophoresis and mass spectrometry. About 3600 bovine mRNAs wereidentified by RNAseq. As expected, most mRNAs were truncated with a biastoward enrichment at the 3′ end. However, 107 mRNAs contained an ATGstart codon, making them putative candidates for translation. Thirteenof the 107 mRNAs encode amino acid sequences not present in their humanorthologs, making them candidates for eliciting an immune response.Seventy-two bovine proteins were identified by RRL and massspectrometry. This shows that mRNAs in bovine milk exosomes aretranslatable into protein which indicates that endogenous mRNA presentin a disclosed bovine milk exosome is translated into protein by amammalian subject, affording the opportunity for treatment of disease oraffecting a physiologic condition in the mammalian subject, such as anyof the conditions discussed herein.

Example 6: Bovine Milk Exosomes and their Corresponding Cargo haveVarious Physiological Functions

A. Depletion of Dietary microRNAs from Cow's Milk Causes an Increase ofPurine Metabolites in Human Body Fluids and Mouse Livers

Previous studies described herein demonstrate that exosomes and theircargo are transported into cells via endocytosis and delivered toperipheral issues. The studies described below show that exosomes andtheir cargo have physiological effect. For instance, dietary depletionof milk miRNAs causes aberrant (an increase in) purine metabolites inmouse tissues and human body fluids. A reversed-phased HPLC method wasdeveloped to quantify eight purine metabolites in a comprehensiveanalysis of purine metabolism.

(i) Exo− and Exo+ Diet

C57BL/6 mice were obtained from Jackson Labs Mice were fed AIN-93G-baseddiets, defined by their content of bovine milk exosomes and their RNAcargos: exosome RNA-depleted (Exo−) versus exosome RNA-sufficient(Exo+). The composition of the two diets is identical, including vesiclecount, except that the milk exosomes in the Exo− diet were depleted ofRNAs by sonication and incubation. Mice were housed in groups of fourmice per cage, separated by sex. Both males and females were studied.True randomization of group assignment was achieved by labeling micewith numbers and randomly assigning numbers to groups. At timedintervals mice were sampled from different cages to avoid cage effects,and euthanized for sample collection.

(ii) Purine Metabolite Study

In a first screen, C57BL/6 mice were fed a diet depleted of miRNAs(encapsulated in exosomes) from age 3 to 7 weeks [denoted Exosome (E)Minus, Exo−], whereas controls were fed a miRNA-sufficient diet (Exo+).Mouse liver metabolites were analyzed using non-targeted LC/MS-MS, andtargeted enzymatic assays for purine metabolites. Subsequently, purinemetabolites in urine and plasma from human dairy avoiders and dairyconsumers was assessed. The statistical significance of differences wasassessed by t-test.

Principal Components Analysis (PCA), a dendrogram performed withhierarchical clustering, Variable Important in Projection (VIP) scores,and a heat map analysis consistently indicated that concentrations ofpurine metabolites were higher in livers of Exo− fed mice than in E+ fedmice, and that purine metabolites were affected by milk miRNA intake toa greater extent than any of the other metabolites that were identified(p<0.01; n=5). As for effect size, the concentrations of xanthine were16.6±3.3 μM and 10.1±1.0 μM in the livers of Exo− and Exo+ mice,respectively (p<0.05). Likewise, the concentrations of plasma xanthineand urinary uric acid were 81.6% and 19.3% higher in human dairyavoiders compared with dairy consumers (n=6).

B. Depletion of Dietary microRNAs from Cow's Milk Causes an Decrease inFertility and Fecundity in Mice

Exosome feeding studies suggest that dietary depletion elicits asubstantial decrease in fertility, intrauterine growth and postnatalsurvival. The studies and corresponding results are described below.

(i) Fertility Study

Mice were fed an exosome-depleted diet versus an exosome-sufficient dietas described above. Mice fed an exosome-depleted (Exo−) dietdemonstrated lower fertility than mice fed an exosome-sufficient (Exo+)mice, particularly when both males and females were fed the Exo+ diet.As shown in FIG. 17, 62% to 75% of mating did not result in pregnancy ifat least one parent was fed the Exo− diet, compared with only one(12.5%) failed mating in controls. As shown in FIG. 18, the averagelitter size produced by Exo+/Exo+ fed breeders was twice the size inother groups: 7.6±1.9 pups for Exo+ fed males/Exo+ fed females, 3.6±3.2for Exo+ fed males/Exo− fed females, 3.5±3.0 for Exo− fed males/Exo+ fedfemales, and 4.1±3.4 for Exo− fed males/Exo− fed females; P<0.05, n=8).The average birth weight of pups born to dams fed the Exo− diet (Exo+males) was 1.130.11 g compared to 1.300.09 g in Exo+ fed/Exo+ fedbreeders (P<0.05). MicroRNA depletion also impaired survival of littersto weaning (3 weeks) if dams were continued on the previous diets. Asshown in FIG. 19, there was 100% survival of litters in Exo+/Exo+ fedbreeders, zero survival in Exo+/Exo− breeders and Exo−/Exo+ breeders,and one surviving litter in Exo−/Exo− breeders.

Gene expression analysis and purine feeding studies are used to identifythe step(s) in purine metabolism targeted by milk miRNAs. The hepaticconcentrations of purine metabolites were 65% higher in Exo− fed femalescompared to Exo+ fed females (16.6±3.3 vs. 10.1±1.0 μmol/L xanthine;P<0.05, n=5), indicating that loss of the positive effects fromendogenous miRNAs leads to lowered fecundity. Dietary depletion of milkmiRNAs causes an increase in purine metabolites in mouse tissues anddecreases in fecundity and fertility.

C. Dietary Depletion of microRNAs from Bovine Milk Exosomes ElicitsChanges in Amino Acid Metabolism in Mice

(i) The Diet and Study

C57BL/6 mice, age 3 weeks, were fed an exosome-depleted (Exo−)AIN93G-based diet for four weeks; controls were fed anexosome-sufficient (Exo+) diet as described above. Livers were harvestedand the hepatic metabolome was assessed by non-targeted LC/MS-MS and bypeak intensity analysis; the hepatic transcriptome was assessed byRNAseq using an Illumina HiSeq2500 platform. A second cohort of mice wasfed Exo− or Exo+ diets for 4-6 weeks for subsequent analysis of gripstrength, respiratory exchange ratio (RER) and feeding and activitypatterns; skeletal muscle samples are currently analyzed by RNAseq.Statistical significance was assessed using unpaired, two-tailed t-test.

(ii) Altered Amino Acid Metabolism

Bovine milk exosomes alter amino acid metabolism in C57BL/6 mice.Hepatic concentrations of amino acids were up to 1800% higher in micefed the Exo− diet than in mice fed the Exo+ diet (control) N=8, *p<0.05vs. Exo+. (FIGS. 20A-E). FIG. 20A shows the abundance of leucine in micefed the Exo− versus Exo+ diet. FIG. 20B shows the abundance ofphenylalanine in mice fed the Exo− versus Exo+ diet. FIG. 20C shows theabundance of alanine in mice fed the Exo− versus Exo+ diet. FIG. 20Dshows the abundance of leucine-lysine dipeptide metabolite in mice fedthe Exo− versus Exo+ diet. FIG. 20E shows the abundance ofglutamyl-isoleucine dipeptide metabolite in mice fed the Exo− versusExo+ diet.

FIGS. 21A and 21B show the mRNA expression of branched chain amino acid(BCAA) transporters 1 (cytoplasm) (BCAT1) and 2 (mitochondria) (BCAT2)in livers of C57BL/6 mice fed an Exo− or Exo+(control) diet for 4 weeks.N=8, *p<0.05 vs. Exo+. The mRNA expression of branched chain amino acid(BCAA) transporters 1 (cytoplasm; BCAT1; FIG. 20A) and 2 (mitochondria,BCAT2; FIG. 20B) was greater in mice fed the Exo− diet compared to micefed the Exo+ diet (n.s. for BCAT2). The respiratory exchange ratio (RER)was not affected by feeding, whereas grip strength was a moderate 5%higher in Exo+vs. Exo-fed females after only 4 weeks of feeding (n.s.;not shown). The trend toward a greater grip strength was not caused bydifferences in food and water consumption or physical activity, whichwere not significantly different between treatment groups in males andfemales.

D. Depletion of Dietary microRNAs in Bovine Milk Exosomes ImpairsSensorimotor Gating and Spatial Learning in Mice

C57BL/6 mice were fed AIN-93G based, bovine milk exosome-defined dietsfor up to 20 weeks. Exosome-depleted diets are denoted Exo− or E−;exosome-sufficient controls are denoted Exo+ or E+. Spatial learning andmemory were assessed using the Barnes maze and Morris water maze.Sensorimotor gating was assessed using an acoustic startle response(ASR) system. The statistical significance of differences was assessedby unpaired, two-tailed t-test.

The time needed to locate the escape hole in the Barnes maze increasedby up to 130% in mice fed the Exo− diet compared to mice fed the Exo+diets (controls) (FIG. 22). Likewise, the time needed to locate andreach the submerged escape platform in the Morris water maze wassignificantly greater in female mice fed the Exo− diet than femalecontrols fed the Exo+ diet (FIG. 23). Prepulse inhibition (PPI) of theASR is a measure of sensorimotor gating and was significantly lower infemale mice fed the Exo− diet than in female controls fed the Exo+ diet(FIG. 24). Diet did not affect PPI in male mice (p>0.05). These studiesshow that bovine milk exosomes improve sensorimotor gating and cognitiveperformance in mice.

Example 7. Bovine Milk Exosomes and their Corresponding Cargo have anEffect on the Mammalian Microbiome A. A Diet Defined by its Content ofBovine Milk Exosomes Alters the Composition of the Intestinal Microbiomein C57BL16 Mice

Exosomes play important roles in cell-to-cell communication, facilitatedby the transfer of exosome cargos such as RNAs, proteins and lipids fromdonor cells to recipient cells. Bacteria communicate with theirenvironment through exosome-like vesicles. Although dietary exosomes inbovine milk are bioavailable, a fraction of milk exosomes reaches thelarge intestine in mice.

The results described here suggest that exosomes in bovine milk changethe composition of the gut microbiome, which is associated with changesin the hepatic transcriptome in mice. Several different studies wereperformed that demonstrate that bovine milk exosomes alter certainmicrobial taxa in the gut microbiome in mice.

The alteration in microbial communities in non-bovine species indicatethat exosomes and their cargos participate in the crosstalk betweenbacterial and animal kingdoms. First, prokaryotic and eukaryoticmicrobes communicate with their environment through exosome-likevesicles. This observation includes gram-positive bacteria, which usevesicles for communication despite the cell wall posing a barrier forvesicle transport. Viruses may participate in exosome signaling throughhijacking and modifying exosomes. Second, up to 20% and 40% of RNAsequence reads in plasma from healthy adults map to bacterial and fungalgenomes, respectively. Third, evidence suggests that orallyadministered, fluorophore-labeled exosomes from bovine milk aredelivered to peripheral tissues. These findings are largely consistentwith the studies suggesting that endogenously and exogenously labeledmilk exosomes accumulate in liver and spleen, but that a considerablefraction of orally administered exosomes escapes absorption and reachesthe large intestine.

(i) Exosome-Depleted (E−) AIN93G-Based Diet Versus Exosome-Sufficient(E+) Diet (3-42 Weeks)

In one study, C57BL/6 mice, age 3 weeks, were fed an exosome-depleted(Exo−) AIN93G-based diet for up to 42 weeks; controls were fed anexosome-sufficient (Exo+) diet. At timed intervals (age 7, 15, 45weeks), cohorts of mice were euthanized and colon content was flashfrozen in liquid nitrogen for subsequent analysis of gut microbiota by16S rRNA gene sequencing of the V4 region using Illumina's MiSeqplatform. Microbial sequences were clustered into Operational TaxonomicUnits (OTUs). High-throughput bacterial 16S rRNA gene sequencingfollowed by clustering of short sequences into operational taxonomicunits (OTUs) is widely used for microbiome profiling. Non-parametrictest was used for statistical analysis.

Depending on sex and age, a total of 51 OTUs were differentiallyabundant between treatment groups; See FIGS. 25-26 for a heat map of thetop 18 OTUs (P<0.05 for age 15 and 45 weeks). For example, the relativeabundance of Firmicute classes Clostridia (Ruminococcaceae) andVerrucomicrobia classes Verrucomicrobiae (Muciniphila) were greater inmice fed E− diet compared with E+ diet at age 15 weeks, whereas therelative abundance of Firmicute classes Clostridia (Clostridiales) wasless in mice fed E− diet compared with E+ diet at age 45 weeks.

(ii) Exosome/RNA-Depleted (ERD) Diet Versus Exosome/RNA-Sufficient (ERS)Diet (3-47 Weeks)

Dietary bovine milk exosomes and their RNA cargos elicited changes inthe composition of the intestinal microbiome and the hepatictranscriptome in C57BL/6 Mice. C57BL/6 mice were fed AIN-93G diets,defined by their content of bovine milk exosomes and RNA cargos:exosome/RNA-depleted (ERD) versus exosome/RNA-sufficient (ERS) diets.Feeding was initiated at age three weeks and continued through age 47weeks, and cecum content and liver samples were collected at ages 7, 15and 47 weeks. 16S rRNA gene sequencing and whole transcriptomesequencing were used to profile microbial communities in the cecum andtranscripts in the liver, respectively, using multivariate protocols.

The dietary intake of exosomes and age, and to a lesser extent sex, hadsignificant effects on the microbial communities in the cecum. At thephyla level, the abundance of Verrucomicrobia was greater in mice fedthe ERD diet compared to the ERS diet, and the abundance of bothFirmicutes and Tenericutes was less in mice fed the ERD compared to theERS at age 47 weeks. At the family level, the abundance ofRuminococcaceae and Lachnospiraceae was greater in males and an unnamedfamily from the order of Clostridiales was smaller in females fed theERS diet compared to the ERD diet at age 7 weeks. Exosome feedingaltered the abundance of 543 operational taxonomic units; diet effectswere particularly strong in the Lachnospiraceae, Ruminococcaceae and theVerrucomicrobiaceae families.

(a) Mouse Feeding Studies

C57BL/6 mice were obtained from Jackson Labs. (stock number 000664) atage three weeks when dietary treatment was initiated. Mice were fedAIN-93G-based diets, defined by their content of bovine milk exosomesand their RNA cargos, exosome RNA-depleted (ERD) versus exosomeRNA-sufficient (ERS). The composition of the two diets is identical,including vesicle count, except that the milk exosomes in the ERD dietwere depleted of RNAs by sonication and incubation. Mice were housed ingroups of four mice per cage, separated by sex. Both males and femaleswere studied. True randomization of group assignment was achieved bylabeling mice with numbers and randomly assigning numbers to groups. Attimed intervals (ages 7, 15 and 47 weeks), mice were sampled fromdifferent cages to avoid cage effects, and euthanized for samplecollection (N=8 for each sex and age). Cecum content was collected,flash frozen in liquid nitrogen and stored at −80° C.; livers werecollected and flash frozen in liquid nitrogen and stored at −80° C. Thestudy was approved by the Institutional Animal Care and Use Committee atthe University of Nebraska-Lincoln (protocol 1229).

(b) Analysis of Microbial Communities

Cecum content was extracted and DNA was purified using the PowerSoil DNAIsolation Kit (Mo Bio Laboratories Inc., Carlsbad, Calif., USA)following the manufacturer's instructions. DNA purity and integrity wereconfirmed by using the 260-to-280 nm ratio (Nanodrop ND-1000, NanodropTechnologies, Wilmington, Del., USA) and agarose gel (0.8%)electrophoresis. The V4 region in the 16S rRNA gene was amplified andsequenced as described previously. The sequencing reads were qualityfiltered and analyzed as described previously. Briefly, contigs weregenerated from paired-end reads and were screened using MOTHUR v.1.38.1to exclude low quality sequences and reads containing ambiguous bases orhomopolymers longer than 8 bp. Additionally, the resulting reads weretrimmed to only retain reads between 245 base pairs (bp) and 275 bp. TheUPARSE pipeline (USEARCH v7.0.1090) was then used to clusterquality-filtered sequences into operational taxonomic units (OTUs) at97% identity, after removal of chimeras using UCHIME. ChimeraSlayergold.fa was used as the reference database for chimera detection.Sequence alignment was performed using the SILVA v123 reference and wasused to build a phylogenetic tree using Clearcut. Taxonomy assignment(Greengenes database: gg_13_8_otus) was performed using QIIME v.1.9.1.Eighty-two samples with an average read count of 40,468 reads and arange of 4,112-144,788 reads were used for downstream analysis. Alphadiversity metrics were used to evaluate richness (Chao1), diversity(Shannon-Weiner index), and coverage (Good's coverage). Rarefactioncurves were constructed using Chao1 values. A core measurable microbiomewas identified based on factors diet, sex, and age. The core measurablemicrobiome was defined as the group of OTUs that are present in at least80% of the samples within each factor. Differences in bacterialcommunities were assessed using permutational multivariate analysis ofvariance (PERMANOVA) utilizing the weighted UniFrac distance matrix.Additionally, the weighted UniFrac distance matrix was used forprincipal coordinate analysis (PCoA). The Linear Discriminant Analysisof Effect Size (LefSe) algorithm with default parameters was used toidentify OTUs that were differentially abundant in the ERS and ERDfeeding groups at different ages. Sequence data were deposited in theNCBI-BioProject database under accession no. PRJNA413623. Kruskal-Wallissum-rank test was used to identify the significant differences inabundance between groups at P<0.05. The Wilcoxon rank-sum test was usedfor pairwise comparisons at adjusted P<0.05, with the Benjamini andHochberg correction. Differences were considered statisticallysignificant if P<0.05.

Alpha diversity (Table 2; FIG. 27) and Beta diversity (Table 3; FIG. 27)suggested that effects of age on microbial communities werestatistically significant (P<0.05), effects of diet on communitiestrended toward statistical significance (P=0.075) and sex had nosignificant effect on microbial communities (P=0.215).

TABLE 2 Comparison of the Alpha diversity in pools defined by age, dietand sex. Group 1 Group 2 T P Group 1 Croup 2 mean ± SD mean ± SD valuevalue Age 47 Age 15 184 ± 13.9 158 ± 24.8 4.795 0.003 weeks weeks Age 15Age 7 158 ± 24.8 142 ± 19.1 2.475 0.016 weeks weeks Age 47 Age 7 184 ±13.9 142 ± 19.1 9.341 0.0015 weeks weeks ERS ERD 165 ± 27.4 157 ± 23.81.386 0.170 Male Female 159 ± 25.0 164 ± 27.2 −0.838 0.399 SD, Standarddeviation.

TABLE 3 Effect of age, diet and sex on microbial communities in thececum of mice F. Df SumsOfSqs MeanSqs Model R² Pr(>F) Sex 1 0.001 0.0011.341 0.008 0.215 Diet 1 0.002 0.002 2.342 0.014 0.075 Age 2 0.065 0.03240.297 0.484 0.001 Sex:Diet 1 0.001 0.001 1.047 0.006 0.326 Sex:Age 20.002 0.001 1.345 0.016 0.215 Diet:Age 2 0.003 0.002 1.950 0.023 0.064Sex:Diet:Age 2 0.004 0.002 2.376 0.029 0.039 Residuals 70 0.056 0.0010.420 Total 81 0.134 1.000 Df, degree of freedom; SumsOfSqs, sum ofsquares; MeanSqs = SumsOfSqs/df, mean squares; F. Model, F-tests.

Combinatorial effects of independent variables on microbial communitieswere statistically significant for the sex×diet×age interaction andtrended toward significance for the diet×age interaction (Table 3). Thefollowing key observations were made at the phyla level regarding ageeffect (FIG. 28). Among the three most abundant phyla, Bacteroidetesincreased over time and was the second most abundant phylum at age 47weeks (P<0.001). The abundance of Firmicutes remained constant betweenages 7 and 15 weeks, but decreased by about 50% at age 47 weeks.Verrucomicrobia were significantly more abundant in mice fed the ERDdiet than in mice fed the ERS diet (P=0.030). The abundance ofActinobacteria decreased considerably over time and this phylum almostdisappeared in mice aged 47 weeks (P<0.001).

Some of the changes at the phylum level involved age×diet interactions.For example, Firmicutes and Tenericutes were not significantly differentin mice fed ERS and ERD diets at age 7 and 15 weeks (P>0.05), but weresignificantly less abundant in mice fed the ERD diet compared with ERSat age 47 weeks (P=0.046 for Firmicutes and P=0.028 for Tenericutes).Some age×diet interactions were detected at younger ages. For example,the abundance of Actinobacteria was greater (P=0.041) in mice fed theERS diet compared with ERD at age 15 weeks. No significant changes atthe phylum level were detected when sex was used as an individualindependent variable (P>0.05). A total of 19 families were identified by16S rRNA sequencing. Lachnospiraceae, Ruminococcaceae and a family fromthe order Clostridiales were the three most abundant families affectedby age, diet or sex (FIG. 29). Exosome-defined diets altered themicrobial communities at the family level, and some of these effectsdepended on diet×age, diet×sex or diet×age×sex interactions. Forexample, at age 7 weeks, Ruminococcaceae, Lachnospiraceae,Coriobacteriaceae, Lactobacillaceae and Mogibacteriaceae families weresignificantly more abundant in male mice fed the ERS diet compared withmales fed the ERD diet (all five P<0.05). In contrast, the abundance ofa family from the order Clostridiales was significantly less abundant infemale mice fed the ERS diet compared with ERD at age 7 weeks (P=0.010).No significant differences were detected at the family level in femalesand males at age 15 weeks. At age 47 weeks, females fed the ERD dietharbored significantly more Bifidobacteriaceae (P=0.005) and lessLactobacillaceae (P=0.018) than ERS females age 47 weeks. Males fed theERD diet harbored significantly more Mogibacteriaceae compared with ERSmales age 47 weeks (P=0.033).

At the level of OTUs, the consumption of milk exosome-defined diets hada strong effect on microbial communities in the mouse cecum (FIG. 30).Two OTUs from the family of Lachnospiraceae were significantly moreabundant in mice fed the ERS diet compared with ERD-fed mice at age 7weeks (P=0.009 and P=0.044, respectively). At age 15 weeks, OTUs fromthe families Verrucomicrobiaceae and S24-7 increased significantly inabundance and became the predominant OTUs in mice fed the ERD dietcompared to ERS diet (P=0.016 and P=0.021, respectively). The diversityof microbial OTUs continued to increase and peaked at age 47 weeks andthese OTUs are from the same families shown in FIG. 28 in both dietgroups.

B. Bovine Milk Exosomes Alter Microbial Communities in the Murine Cecumwhich Changes Contribute to Changes in the Murine Hepatic Transcriptome

Exosome-dependent changes in microbial communities correlated withchanges in the hepatic transcriptome, e.g., in pathways implicated innon-alcoholic fatty liver disease and oxidative phosphorylation.

(i) Analysis of the Hepatic Transcriptome

Livers from six female mice, age 15 weeks, were used for RNA sequencingstudies (N=3 from each feeding group). Briefly, RNA was extracted usingthe mRNA Seq Sample Prep Kit (Illumina) and shipped on dry ice to thegenomics center of University of Minneapolis, Minn. for sequencinganalysis. RNA quality was assessed using an Agilent Bioanalyzer andabsorbance at 260 and 280 nm. The RNA Integrity Number and the260-to-280 nm ratio was greater than 7 and 1.8, respectively, for allsamples. Libraries were generated using TruSeq Stranded Total RNALibrary Prep Kit and sequenced using the Illumina HiSeq 2500 platformand a paired ends protocol generating reads with a length of 125 bp.Data quality control was performed using FastQC. After removing adaptorsand reads containing ambiguous bases or having average quality scoreless than 30, sequencing reads were aligned to the mouse referencegenome [GRCm38, mm10] using Tophat. Cufflinks was applied to identifythe transcripts and quantify their expression in units of reads perkilobase of exon model per million (RPKM). Cuffdiff was applied toidentify the differentially expressed transcripts between two feedinggroups and only those with equal or more than 2-fold change wereconsidered for the downstream analysis. KEGG pathways were identified byusing clusterProfiler. Raw sequencing data were deposited in theNCBI-BioProject database under accession ID PRJNA400248.

(ii) Correlation Analysis of Microbial Communities and HepaticTranscriptome.

Correlations between the significantly differentially abundant OTUs andthe top 69 significantly differentially expressed genes were calculatedusing the Pearson product-moment correlation and bootstrapping with 1000permutations to calculate the p-values of the correlation scores (QIIMEv.1.9.1). To visualize the microbial features that correlated with geneexpression, a heatmap was generated using R software package 3.3.3 (TheR Foundation). Bray-Curtis dissimilarity was used to calculate thedistance for rows and columns, and average linkage hierarchicalclustering was used to generate dendrograms.

Sixty-nine genes were differentially expressed by at least 2-fold in thelivers of female mice fed the ERS diet compared to ERD females at age 15weeks (P<0.01). Changes in the hepatic transcriptome correlated withchanges in microbial communities in the cecum. The strong correlationbetween OTUs from the family of Lachnospiraceae (phylum Firmicutes) withhepatic transcripts is particularly noteworthy (FIG. 31). For example,Lachnospiraceae OTUs 22, 7 and 40 correlated strongly with theexpression of the differentially expressed genes in the liver of femalemice fed milk exosome-defined diets. Strong correlations were alsoobserved for Ruminococcaceae OTUs 19 and 34, Erysipelotrichaceae OTU 3and five OTUs that could not be assigned to a family (OTUs 10, 18, 30,33 and an unnamed OTU). A full record of correlation and Gene IDs forthe genes are shown in the tables below.

TABLE 4 Genes and Corresponding Gene IDs from FIG. 31 Gene Gene ID Marco8685 (human) 17167 (mouse) Cxc113 10563 (human) 55985 (mouse) Dnajc1256521 (human) 30045 (mouse) Saa3 6290 (human) 20210 (mouse) A2m 2(human) 232345 (mouse) Rpph1 85495 (human) 85029 (mouse) Mup9 100038948(mouse) Pfdn6 10471 (human) 14976 (mouse) Ndufs6 4726 (human) 407785(mouse) Ssna1 8636 (human) 68475 (mouse) Zfp524 66056 (mouse) Gfer 2671(human) 11692 (mouse) Ccdc124 115098 (human) 234388 (mouse) Ndufb2 4708(human) 68198 (mouse) Isoc2b 67441 (mouse) Use1 55850 (human) 67023(mouse) Rpp21 79897 (human) 67676 (mouse) Rnaseh2c 84153 (human) 67209(mouse) Cblc 23624 (human) 80794 (mouse) Nat9 26151 (human) 66176(mouse) Brms1 25855 (human) 107392 (mouse) Crip1 1396 (human) 12925(mouse) Tpgs1 91978 (human) 110012 (mouse) Rnaseh2a 10535 (human) 69724(mouse) Pet100 100131801 (human) 100503890 (mouse) Bcl7c 9274 (human)12055 (mouse) Trappc6a 79090 (human) 67091 (mouse) Ddx49 54555 (human)234374 (mouse) Trappc5 126003 (human) 66682 (mouse) Sssca1 10534 (human)56390 (mouse) Zfp771 244216 (mouse) Timm50 92609 (human) 66525 (mouse)Scnm1 79005 (human) 69269 (mouse) Fars2 10667 (human) 69955 (mouse)Acbd5 91452 (human) 74159 (mouse) Stx4a 6810 (human) 20909 (mouse) Smagp57228 (human) 207818 (mouse) Mrps16 51021 (human) 66242 (mouse) Hspbp123640 (human) 66245 (mouse) Zap70 7535 (human) 22637 (mouse) Cenpm 79019(human) 66570 (mouse) Slc16a11 162515 (human) 21687 (mouse) Ndufa5 4698(human) 68202 (mouse) Epb4.1/4aos 269587 (mouse) Tmem223 79064 (human)66836 (mouse) Lgals1 3956 (human) 16852 (mouse) Rps19bp1 91582 (human)66538 (mouse) Mrps21 54460 (human) 66292 (mouse) Uqcc3 790955 (human)107197 (mouse) Mrpl23 6150 (human) 19935 (mouse) Cox7a1 1346 (human)12865 (mouse) BC029214 Replaced by Paxx 227622 (mouse) Pmf1 11243(human) 67037 (mouse) Ccdc23 Listed as SVPB 374969 (human) 69216 (mouse)Smim22 440335 (human) 432995 (mouse) Haus7 55559 (human) 73738 (mouse)Cda 978 (human) 72269 (mouse) Tmem238 388564 (human) 664968 (mouse)Bola2 552900 (human) 66162 (mouse) Mup1 17840 (mouse) Mup12 100039054(mouse) Gpihbp1 338328 (human) 68453 (mouse) Nr0b2 8431 (human) 23957(mouse) Mrps18a 55168 (human) 68565 (mouse) Atoh8 84913 (human) 71093(mouse) Nudt1 4521 (human) 17766 (mouse) Gsta4 2941 (human) 14860(mouse) Mup21 381531 (mouse)

TABLE 5 Microbiome and Transcriptome Correlation Results. No No No NoAssigned Assigned Assigned Assigned gene Lactobacillaceae FamilyErysipelotrichaceae_1 Lachnospiraceae_7 Family_4 Family_7 Family_10 A2m0.01 0.62 0.77 0.95 0.32 0.60 0.97 Acbd6 0.99 0.06 1.00 0.17 0.65 0.670.12 Atoh8 0.95 0.05 0.81 0.20 0.43 0.54 0.12 BC029214 0.93 0.13 1.000.21 0.65 0.72 0.14 Bcl7c 0.98 0.11 0.98 0.25 0.74 0.68 0.12 Bola2 0.910.13 0.94 0.18 0.76 0.76 0.15 Brms1 0.94 0.06 1.00 0.20 0.68 0.75 0.22Cblc 0.99 0.10 1.00 0.23 0.69 0.74 0.16 Ccdc124 0.88 0.07 1.00 0.15 0.760.80 0.14 Ccdc23 0.84 0.14 0.98 0.23 0.58 0.74 0.21 Cda 0.98 0.09 0.970.03 0.57 0.64 0.07 Cenpm 0.95 0.03 1.00 0.25 0.62 0.61 0.10 Cox7a1 0.980.08 1.00 0.19 0.65 0.69 0.12 Crip1 0.99 0.08 0.99 0.30 0.71 0.72 0.15Cxcl13 0.16 0.43 0.67 0.41 0.04 0.40 0.64 Ddx49 0.91 0.13 0.97 0.00 0.650.68 0.14 Dnajc12 0.16 0.29 0.38 0.27 0.05 0.54 0.33 Epb4.1l4aos 0.920.10 0.98 0.23 0.67 0.63 0.15 Fars2 0.97 0.06 1.00 0.14 0.69 0.71 0.06Gfer 0.95 0.08 0.95 0.13 0.62 0.74 0.15 Gpihbp1 0.81 0.02 0.99 0.06 0.570.76 0.06 Gsta4 0.78 0.09 0.98 0.13 0.54 0.60 0.14 Haus7 0.88 0.13 1.000.13 0.60 0.68 0.17 Hspbp1 0.98 0.08 0.98 0.13 0.60 0.70 0.08 Isoc2b0.99 0.07 0.94 0.14 0.68 0.70 0.17 Lgals1 1.00 0.11 0.98 0.24 0.72 0.770.08 Marco 0.14 0.44 0.71 0.49 0.01 0.38 0.44 Mrpl23 1.00 0.07 0.99 0.160.64 0.71 0.14 Mrps16 0.96 0.06 1.00 0.20 0.53 0.74 0.12 Mrps18a 0.980.08 0.83 0.08 0.42 0.57 0.21 Mrps21 0.96 0.06 0.99 0.23 0.69 0.70 0.18Mup12 0.97 0.07 0.98 0.23 0.72 0.88 0.19 Mup1 0.98 0.03 1.00 0.16 0.680.75 0.17 Mup21 0.96 0.19 0.08 0.91 0.07 0.00 0.57 Mup9 1.00 0.04 0.510.19 0.63 0.91 0.11 Nat9 0.96 0.11 1.00 0.24 0.70 0.72 0.17 Ndufa5 0.970.09 0.97 0.30 0.52 0.71 0.15 Ndufb2 0.97 0.13 0.99 0.09 0.71 0.69 0.12Ndufs6 0.97 0.12 1.00 0.14 0.62 0.72 0.18 Nr0b2 0.97 0.09 0.79 0.02 0.440.52 0.18 Nudt1 0.95 0.14 0.97 0.21 0.48 0.52 0.09 Pet100 0.92 0.13 0.980.21 0.72 0.70 0.13 Pfdn6 1.00 0.12 0.98 0.15 0.66 0.76 0.18 Pmf1 0.950.09 0.99 0.22 0.55 0.71 0.14 Rnaseh2a 0.86 0.13 1.00 0.18 0.71 0.660.12 Rnaseh2c 0.97 0.08 1.00 0.17 0.57 0.66 0.13 Rpp21 0.96 0.05 1.000.24 0.57 0.78 0.14 Rpph1 0.79 0.17 0.80 0.18 0.74 0.93 0.20 Rps19bp10.98 0.11 0.99 0.26 0.64 0.71 0.11 Saa3 0.05 0.52 0.74 0.99 0.23 0.490.89 Scnm1 0.97 0.16 1.00 0.18 0.65 0.69 0.14 Slc16a11 0.97 0.07 1.000.21 0.48 0.65 0.13 Smagp 0.99 0.11 1.00 0.24 0.54 0.61 0.14 Smim22 0.950.14 1.00 0.19 0.75 0.70 0.13 Ssna1 0.92 0.11 1.00 0.19 0.64 0.69 0.15Sssca1 0.87 0.11 0.96 0.15 0.65 0.73 0.13 Stx4a 1.00 0.13 1.00 0.21 0.590.63 0.15 Timm50 0.99 0.09 1.00 0.12 0.67 0.76 0.17 Tmem223 0.96 0.091.00 0.17 0.77 0.71 0.07 Tmem238 0.92 0.10 0.97 0.19 0.74 0.67 0.17Tpgs1 0.91 0.15 1.00 0.16 0.63 0.65 0.19 Trappc5 0.90 0.11 1.00 0.110.75 0.69 0.16 Trappc6a 0.90 0.13 0.97 0.03 0.64 0.68 0.15 Uqcc3 1.000.09 1.00 0.26 0.67 0.77 0.18 Use1 0.93 0.17 0.98 0.14 0.57 0.75 0.13Zap70 0.99 0.03 0.99 0.21 0.61 0.69 0.09 Zfp524 0.95 0.08 1.00 0.16 0.700.76 0.17 Zfp771 0.88 0.10 1.00 0.25 0.73 0.62 0.10 No No No No AssignedAssigned Assigned Assigned gene Lachnospiraceae_22 Family_17 Family_18Erysipelotrichaceae_3 Lachnospiraceae_28 Family_21 Family_22 A2m 0.740.00 0.63 0.77 0.89 0.41 0.27 Acbd6 0.72 0.83 0.06 0.15 0.94 0.99 0.97Atoh8 0.65 0.92 0.03 0.13 0.81 0.96 0.86 BC029214 0.64 0.88 0.08 0.110.68 0.98 0.92 Bcl7c 0.59 0.77 0.01 0.16 0.85 0.97 1.00 Bola2 0.58 0.670.05 0.13 0.58 0.91 0.86 Brms1 0.18 0.72 0.02 0.11 0.82 0.98 1.00 Cblc0.13 0.76 0.03 0.17 0.71 1.00 0.99 Ccdc124 0.10 0.64 0.05 0.15 0.83 0.991.00 Ccdc23 0.08 0.75 0.05 0.15 1.00 0.96 1.00 Cda 0.08 0.73 0.02 0.070.88 1.00 0.95 Cenpm 0.08 0.70 0.03 0.07 0.86 1.00 0.99 Cox7a1 0.08 0.880.02 0.07 0.71 0.98 0.99 Crip1 0.08 0.73 0.06 0.11 0.61 0.96 0.97 Cxcl130.07 0.22 0.46 0.25 0.81 0.32 0.27 Ddx49 0.07 0.73 0.05 0.17 0.77 0.940.98 Dnajc12 0.07 0.19 0.22 0.30 0.88 0.35 0.26 Epb4.1l4aos 0.07 0.890.07 0.17 0.79 0.99 0.95 Fars2 0.07 0.77 0.02 0.06 0.95 1.00 0.92 Gfer0.07 0.68 0.11 0.18 0.82 0.98 1.00 Gpihbp1 0.07 0.65 0.03 0.06 0.71 0.970.96 Gsta4 0.07 0.99 0.04 0.16 0.97 0.99 0.78 Haus7 0.07 0.91 0.05 0.070.72 0.90 0.94 Hspbp1 0.06 0.79 0.09 0.05 0.88 0.99 0.97 Isoc2b 0.060.65 0.02 0.19 0.81 1.00 1.00 Lgals1 0.06 0.79 0.02 0.14 0.74 0.98 1.00Marco 0.06 0.14 0.45 0.44 0.70 0.33 0.16 Mrpl23 0.06 0.78 0.05 0.11 0.730.95 0.99 Mrps16 0.06 0.75 0.07 0.14 0.94 1.00 0.95 Mrps18a 0.06 0.790.07 0.08 0.87 0.98 0.96 Mrps21 0.06 0.75 0.04 0.11 0.89 0.98 0.99 Mup120.06 0.78 0.01 0.11 0.62 0.96 0.98 Mup1 0.05 0.74 0.04 0.08 0.56 0.990.98 Mup21 0.05 0.82 0.37 0.82 0.05 0.02 0.07 Mup9 0.05 0.77 0.05 0.130.55 0.96 0.99 Nat9 0.05 0.65 0.06 0.17 0.70 1.00 0.97 Ndufa5 0.05 0.850.01 0.13 0.83 0.97 0.99 Ndufb2 0.05 0.78 0.04 0.13 0.82 0.99 1.00Ndufs6 0.05 0.75 0.03 0.12 0.85 0.99 1.00 Nr0b2 0.05 0.82 0.03 0.15 0.750.98 0.98 Nudt1 0.04 0.83 0.01 0.11 0.99 0.98 0.94 Pet100 0.04 0.72 0.030.16 0.82 0.91 0.97 Pfdn6 0.04 0.72 0.06 0.10 0.82 1.00 1.00 Pmf1 0.040.93 0.06 0.09 0.79 0.99 0.94 Rnaseh2a 0.04 0.67 0.04 0.13 0.81 0.980.95 Rnaseh2c 0.04 0.79 0.02 0.17 0.69 0.99 0.98 Rpp21 0.04 0.76 0.030.14 0.87 0.98 1.00 Rpph1 0.04 0.64 0.05 0.22 0.53 0.88 0.85 Rps19bp10.04 0.79 0.05 0.14 0.91 0.98 1.00 Saa3 0.04 0.00 0.55 0.76 0.94 0.380.19 Scnm1 0.03 0.70 0.03 0.10 0.95 0.99 0.96 Slc16a11 0.03 0.88 0.020.16 0.81 1.00 0.92 Smagp 0.03 0.70 0.03 0.14 0.93 0.98 0.95 Smim22 0.030.67 0.04 0.09 0.61 0.97 0.97 Ssna1 0.02 0.69 0.02 0.09 0.79 0.99 1.00Sssca1 0.02 0.76 0.07 0.15 0.70 0.95 0.95 Stx4a 0.02 0.74 0.05 0.05 1.000.97 0.98 Timm50 0.02 0.79 0.00 0.15 0.90 0.96 0.99 Tmem223 0.02 0.800.04 0.14 0.80 0.99 1.00 Tmem238 0.02 0.66 0.02 0.17 0.65 0.96 0.92Tpgs1 0.02 0.73 0.02 0.26 0.81 0.97 0.96 Trappc5 0.02 0.66 0.05 0.200.70 0.95 0.95 Trappc6a 0.02 0.66 0.02 0.13 0.86 0.97 1.00 Uqcc3 0.010.84 0.00 0.13 0.82 0.99 1.00 Use1 0.01 0.72 0.01 0.11 0.74 0.97 0.98Zap70 0.01 0.77 0.01 0.08 0.96 1.00 0.92 Zfp524 0.01 0.72 0.00 0.10 0.751.00 0.91 Zfp771 0.00 0.63 0.02 0.13 0.76 0.96 0.97 No Assigned geneRuminococcaceae_19 Clostridiaceae_2 Lachnospiraceae_40 Family_30Lachnospiraceae_43 Lachnospiraceae_44 A2m 0.78 0.17 0.96 0.80 0.12 0.04Acbd6 0.11 0.23 0.18 0.17 0.41 0.65 Atoh8 0.19 0.24 0.18 0.07 0.35 0.54BC029214 0.14 0.24 0.24 0.32 0.48 0.72 Bcl7c 0.13 0.24 0.21 0.17 0.550.86 Bola2 0.19 0.52 0.23 0.23 0.73 0.91 Brms1 0.27 0.28 0.23 0.17 0.700.81 Cblc 0.09 0.26 0.20 0.20 0.50 0.78 Ccdc124 0.16 0.29 0.20 0.09 0.640.84 Ccdc23 0.23 0.39 0.22 0.08 0.48 0.80 Cda 0.04 0.28 0.02 0.09 0.560.83 Cenpm 0.10 0.27 0.20 0.05 0.59 0.75 Cox7a1 0.16 0.22 0.33 0.22 0.490.75 Crip1 0.05 0.14 0.15 0.10 0.60 0.73 Cxcl13 0.16 0.15 0.36 0.53 0.060.17 Ddx49 0.19 0.31 0.02 0.11 0.66 0.86 Dnajc12 0.37 0.09 0.19 0.560.13 0.23 Epb4.1l4aos 0.17 0.27 0.22 0.14 0.35 0.64 Fars2 0.21 0.28 0.200.11 0.45 0.64 Gfer 0.18 0.33 0.18 0.09 0.63 0.79 Gpihbp1 0.16 0.49 0.030.22 0.94 0.92 Gsta4 0.15 0.28 0.10 0.25 0.35 0.35 Haus7 0.15 0.28 0.070.14 0.39 0.66 Hspbp1 0.15 0.29 0.20 0.12 0.59 0.74 Isoc2b 0.15 0.180.13 0.13 0.66 0.75 Lgals1 0.10 0.19 0.16 0.18 0.52 0.71 Marco 0.41 0.140.65 0.67 0.11 0.21 Mrpl23 0.14 0.17 0.18 0.20 0.54 0.75 Mrps16 0.140.20 0.23 0.15 0.56 0.74 Mrps18a 0.09 0.29 0.05 0.08 0.31 0.60 Mrps210.12 0.30 0.18 0.07 0.50 0.77 Mup12 0.13 0.33 0.19 0.35 0.81 0.83 Mup10.17 0.26 0.12 0.25 0.74 0.79 Mup21 0.66 0.41 0.87 0.18 0.72 0.39 Mup90.01 0.40 0.21 0.41 0.80 0.86 Nat9 0.14 0.15 0.20 0.12 0.72 0.83 Ndufa50.19 0.29 0.17 0.16 0.46 0.72 Ndufb2 0.13 0.20 0.02 0.21 0.66 0.68Ndufs6 0.11 0.23 0.19 0.08 0.60 0.67 Nr0b2 0.14 0.29 0.01 0.11 0.27 0.69Nudt1 0.14 0.17 0.21 0.13 0.35 0.65 Pet100 0.21 0.34 0.13 0.14 0.56 0.81Pfdn6 0.14 0.26 0.15 0.13 0.53 0.73 Pmf1 0.13 0.29 0.26 0.22 0.48 0.65Rnaseh2a 0.23 0.54 0.18 0.13 0.66 0.82 Rnaseh2c 0.08 0.23 0.20 0.14 0.610.81 Rpp21 0.12 0.19 0.15 0.16 0.59 0.83 Rpph1 0.18 0.69 0.12 0.32 0.940.97 Rps19bp1 0.10 0.24 0.21 0.15 0.49 0.77 Saa3 0.65 0.23 0.99 0.770.13 0.06 Scnm1 0.17 0.25 0.12 0.12 0.40 0.61 Slc16a11 0.11 0.27 0.140.18 0.46 0.74 Smagp 0.16 0.26 0.26 0.10 0.42 0.64 Smim22 0.18 0.32 0.170.16 0.38 0.70 Ssna1 0.06 0.29 0.19 0.09 0.57 0.69 Sssca1 0.27 0.37 0.110.15 0.65 0.79 Stx4a 0.15 0.23 0.23 0.10 0.39 0.65 Timm50 0.17 0.25 0.180.08 0.37 0.64 Tmem223 0.25 0.23 0.15 0.14 0.38 0.72 Tmem238 0.09 0.260.22 0.29 0.87 0.87 Tpgs1 0.22 0.32 0.23 0.07 0.61 0.83 Trappc5 0.260.38 0.13 0.12 0.60 0.77 Trappc6a 0.19 0.33 0.07 0.10 0.57 0.86 Uqcc30.12 0.15 0.22 0.12 0.53 0.77 Use1 0.09 0.17 0.16 0.19 0.60 0.80 Zap700.07 0.31 0.18 0.09 0.61 0.73 Zfp524 0.18 0.25 0.22 0.12 0.59 0.68Zfp771 0.13 0.30 0.24 0.19 0.64 0.69 No Assigned gene Ruminococcaceae_34Family_33 Lachnospiraceae_46 Lachnospiraceae_47 A2m 0.89 0.66 0.65 0.66Acbd6 0.01 0.16 0.74 0.93 Atoh8 0.09 0.04 1.00 1.00 BC029214 0.11 0.230.72 0.94 Bcl7c 0.01 0.20 0.79 1.00 Bola2 0.03 0.37 0.72 0.91 Brms1 0.070.32 0.68 1.00 Cblc 0.10 0.22 0.63 1.00 Ccdc124 0.04 0.15 0.63 0.86Ccdc23 0.10 0.22 0.80 0.97 Cda 0.07 0.08 0.81 0.90 Cenpm 0.03 0.13 0.751.00 Cox7a1 0.08 0.20 0.74 0.98 Crip1 0.07 0.32 0.68 1.00 Cxcl13 0.600.30 0.55 0.80 Ddx49 0.09 0.21 0.70 1.00 Dnajc12 0.44 0.18 0.63 0.45Epb4.1l4aos 0.11 0.15 0.83 1.00 Fars2 0.06 0.11 0.77 0.98 Gfer 0.07 0.100.72 0.87 Gpihbp1 0.10 0.20 0.79 1.00 Gsta4 0.09 0.10 0.97 0.87 Haus70.08 0.20 0.72 1.00 Hspbp1 0.11 0.10 0.74 1.00 Isoc2b 0.07 0.08 0.841.00 Lgals1 0.08 0.12 0.74 0.93 Marco 0.70 0.34 0.51 0.80 Mrpl23 0.110.21 0.71 0.96 Mrps16 0.09 0.09 0.73 1.00 Mrps18a 0.08 0.10 1.00 1.00Mrps21 0.09 0.18 0.75 0.96 Mup12 0.06 0.27 0.64 0.91 Mup1 0.13 0.29 0.810.96 Mup21 0.27 0.04 0.02 0.08 Mup9 0.24 0.31 0.51 0.47 Nat9 0.11 0.210.66 0.93 Ndufa5 0.01 0.24 0.83 0.98 Ndufb2 0.10 0.14 0.80 0.98 Ndufs60.05 0.10 0.75 0.75 Nr0b2 0.05 0.04 0.96 0.91 Nudt1 0.05 0.09 0.84 0.91Pet100 0.06 0.16 0.74 1.00 Pfdn6 0.08 0.09 0.79 0.86 Pmf1 0.08 0.19 0.740.89 Rnaseh2a 0.06 0.25 0.76 1.00 Rnaseh2c 0.07 0.27 0.73 1.00 Rpp210.01 0.18 0.68 0.94 Rpph1 0.17 0.49 0.53 0.76 Rps19bp1 0.15 0.07 0.820.99 Saa3 1.00 0.83 0.63 0.76 Scnm1 0.11 0.06 0.75 0.97 Slc16a11 0.090.14 0.77 1.00 Smagp 0.12 0.07 0.76 1.00 Smim22 0.08 0.24 0.65 0.92Ssna1 0.16 0.13 0.76 0.86 Sssca1 0.10 0.26 0.74 0.95 Stx4a 0.08 0.180.81 1.00 Timm50 0.12 0.10 0.76 0.97 Tmem223 0.11 0.16 0.80 0.97 Tmem2380.09 0.41 0.65 0.98 Tpgs1 0.10 0.14 0.77 1.00 Trappc5 0.10 0.31 0.641.00 Trappc6a 0.08 0.14 0.75 0.91 Uqcc3 0.05 0.19 0.83 0.98 Use1 0.110.24 0.70 0.94 Zap70 0.18 0.08 0.81 1.00 Zfp524 0.05 0.27 0.81 0.86Zfp771 0.14 0.23 0.75 1.00

These 69 differentially expressed genes are implicated in many diseases,such as Huntington's disease, Alzheimer's disease, Non-alcoholic fattyliver disease (NAFLD) and Parkinson's disease (FIG. 32) as determined byKEGG pathway analysis for metabolic functions that are enriched in thehepatic transcriptome in female mice, age 15 weeks fed exosomeRNA-sufficient (ERS) or exosome RNA-depleted (ERD).

This study provides strong evidence that dietary exosomes and their RNAcargos, at least those in milk, are responsible for some of the effectsof diet on the gut microbiome. This study indicates that dietary RNAselicit changes in gene expression across kingdoms, in this case animalsand bacteria. This concept is based on a report suggesting that MIR-168ain rice is bioavailable and binds to the mRNA coding for low-densitylipoprotein receptor adaptor protein 1, thereby lowering mRNA expressionin the liver of mice. This study suggests that gut microorganisms mightact as transmitters or amplifiers of dietary exosome signals. Previousreports have suggested that microRNA cargos in dietary exosomes achievetissue concentrations that are too low to elicit biological effects andthat the bioavailability of milk exosomes might be low. Here evidence isprovided that exosome-defined diets alter microbial communities in themurine cecum, and effects are particularly strong if studied at the OTUlevel. Importantly, milk exosomes that escape absorption by mucosalcells may still elicit major biological effects, facilitated by the gutmicrobiome.

C. The Microbial Communities Altered by Milk Exosome-Defined Diets areRelated to Pathological and Physiological Conditions

It is now widely accepted that prokaryotic and eukaryotic microbescommunicate with their environment through exosome-like vesicles.Changes in microbial communities are paralleled by changes in theproduction of microbial metabolites, which may transmit and amplify milkexosome signals. These studies provide evidence that milkexosome-dependent changes in microbial communities can explain changesobserved in the hepatic transcriptome in mice.

The microbial communities altered by milk exosome-defined diets arerelated to pathological and physiological conditions, as evidenced bythe following examples. A loss of Lachnospiraceae and Ruminococcaceaeand a gain in Enterobacteriaceae in the ileal mucosa have beenimplicated in inflammatory bowel disease. The ratio of Firmicutes andBacteroidetes is greater in obese compared with lean subjects. See,e.g., Daulatzai M. A., Mol. Genet. Med. S1:005 10.4172/1747-0862.S1-005.

Dysbiosis in the gut microbiome may cause liver disease due to microbialmetabolites altering the metabolism in hepatic cells via innate immunereceptors, e.g., a decreased abundance of Ruminococcaceae andEscherichia has been linked with non-alcoholic fatty liver disease. See,e.g., Adams, L. A. et al., Curr. Hepatol. Rep. 15 96-102.10.1007/s11901-016-0299-5. Inflammatory bowel disease, obesity andnon-alcoholic fatty liver disease are major health concerns in theUnited States.

Gut microbiota are likely not the only amplifiers of exosome and RNAcargo signals. It has been proposed that microRNAs elicit biologicaleffects through binding to Toll-like receptors or by surfaceantigen-mediated delivery of exosomes to immune cells to create anexosome-rich microenvironment; mere exosome-cell surface interactionsmight also alter cell signaling pathways.

This study provides strong evidence that milk exosomes and their RNAcargos elicit biological effects across species boundaries and evenkingdoms. Of significant importance is the observation that exosomesthat escape intestinal absorption have biological activity, caused byexosome-microbiota interactions.

Example 8: Glycoproteins on the Surface of Milk Exosomes Mediate theUptake of Exosomes into Human and Other Mammalian Cells

Extracellular vesicles (EVs), such as exosomes, carry cargo thatincludes various species of RNA, proteins, and lipids. Encapsulation ofsuch cargos in EVs confers protection against degradation and a pathwayfor cellular uptake by endocytosis. EVs are secreted by donor cells fordelivery to recipient cells, and EV cargo has emerged as an importantmediator in intercellular communication. There are 3 major classes ofEVs, i.e., exosomes, microvesicles, and apoptotic bodies. Exosomes areof particular interest because they are loaded with microRNAs in atargeted, nonstochastic process that involves sorting mechanisms.

The intestinal transport of bovine milk exosomes (and microRNAs) can beassessed using fluorophore-labeled bovine milk exosomes in mammaliancell cultures, such as human epithelial colon adenocarcinoma Caco-2cells, normal rat small intestinal IEC-6 cells, human small intestinalcells (FH), and human umbilical vein endothelial cells (HUVEC).Transport kinetics and mechanisms can be characterized using such assaysas: dose-response studies, inhibitors of vesicle transport, carbohydratecompetitors, proteolysis of surface proteins on cells and exosomes, andtransepithelial transport in transwell plates.

The studies described herein demonstrate that: (1) milk exosomes aretransported into human and mammalian cells in a dose-dependent manner(until saturation); (2) transport occurs via endocytosis; (3) cellsurface glycoproteins play a significant role in the transport ofexosomes; and (4) glycans on surface glycoproteins play a significantrole in the transport of exosomes. Modification of the exosome surfaceglycoproteins and/or glycans can be used not only to regulate the rateand amount of exosome transport (and cargo delivery), but can also beused to direct exosomes to a particular cellular target.

A. Transport of Milk Exosomes into Human and Other Mammalian Cells

Exosomes were isolated from cow's milk using ultracentrifugation asdescribed in Example 1. The identity, purity, and integrity of theisolated exosomes were confirmed using nanoparticle tracker, westernblot, and transmission electron microscopy as described in Example 1.

(i) Temporal Studies

Caco-2 cells and IEC-6 cells were seeded at a density of 20,000 and 7000cells/well, respectively, in 96-well plates, and allowed to adhere for48 h, when cells were 75% confluent. The exosomes were labeled with thefluorophore, FM 4-64 (Molecular Probes). One microliter of a stocksolution of FM 4-64 (5.9 mmol/L) was added to 1 mL of exosome suspensionand incubated for 15 min at 37° C., and excess FM 4-64 was removed byultracentrifugation at 120,000 3 g at 4° C. for 90 min. Transportstudies were conducted using FM4-64-labeled exosomes using 3-110 mg ofexosomal protein/well (Caco-2 cells) or 27-652 mg of exosomalprotein/well (IEC-6 cells) and incubating cells for various periods oftime to assess saturation kinetics; blanks were created using solvent.Assays were calibrated by quantifying the fluorescence of a known massof exosomes labeled with FM 4-64. Exosome uptake was analyzed bymeasuring the cell fluorescence at 515 (excitation) and 640 nm(emission) using a Biotek FLx800 plate reader (BioTek Instru-ments).Fluorescence readings were corrected for cell autofluorescence bysubtracting signals measured in cells incubated with exosome-depletedmedia. Transport kinetics was modeled using the Michaelis-Mentenequation and nonlinear regression; modeling was conducted using GraphPadPrism 6.0 (GraphPad Software). In Caco-2 cells, exosome uptake waslinear for up to 120 min if transport was measured using nonsaturatingsubstrate concentrations (FIG. 33A): y=0.0012×+0.014 (r2=0.97; P<0.05).In IEC-6 cells exosome uptake was linear for only up to 60 min iftransport was measured using nonsaturating substrate concentrations(FIG. 33B): y=0.0033×+0.033 (r2=0.75; P<0.05). Subsequent transportstudies were conducted using incubation times of 8 hours for Caco-2cells (FIG. 34B). Transport of milk exosome uptake was also studied inhuman umbilical vein endothelial cells (HUVECs) human small intestinalcells (FHs cells). FIG. 34A shows separate experiments in whichtransport of milk exosomes in HUVECs was measured over 120 minutes (2hours) (insert for FIG. 34A) and 480 minutes (4 hours) using 20 μgexosome protein/200 μl of media (FIG. 34A). FIG. 34C shows an exosomeuptake study in human small intestinal cells (FHs cells) over the courseof 8 hours.

(ii) Substrate Studies

In both mammalian cells, the uptake of bovine milk exosomes was mediatedby saturable transport mechanisms. Transport kinetics was modeled usingthe Michaelis-Menten equation. Exosome uptake was analyzed by measuringthe cell fluorescence at 515 (excitation) and 640 nm (emission) using aBiotek FLx800 plate reader (BioTek Instruments) as previously describedabove. In Caco-2 cells, Michaelis constant (K_(m)) and maximal transportrate were 55.5+48.6 μg exosomal protein/200 μL medium and 0.08+0.06 ngof exosomal protein/81,750 cells⁻¹/h⁻¹, respectively (r2=0.75; FIG.35A). In IEC-6 cells Km and maximal transport rate were 152+39.5 μg/200μL and 0.14+0.01 ng of exosomal protein/36,375 cells 30⁻¹/min⁻¹,respectively (r2=0.56; FIG. 35B). When the incubation temperature wasdecreased from 37° C. to 4° C., the transport rate decreased from100%+56% to 54%+13% using a substrate concentration of 55.5 μg exosomalprotein/200 μL in Caco-2 cells (P<0.05; n=3). Likewise, when theincubation temperature was decreased from 37° C. to 4° C., the transportrate decreased from 100%+11% to 44%+25% using a substrate concentrationof 153 μg exosomal protein/200 μL in IEC-6 cells (P<0.05; n=3).Subsequent transport studies were conducted using substrateconcentrations of 55 μg/200 mL and 153 μg/200 mL in Caco-2 cells andIEC-6 cells, respectively. FIG. 36A shows saturation kinetics of milkexosome uptake in CaCo2 as a function of substrate concentration at 37°C. (N=3; p<0.05). FIG. 36B shows exosome uptake into human umbilicalvein endothelial cells as a function of substrate concentration at 37°C. FIGS. 37A and 37B show saturation kinetics of milk exosome uptake inhuman small intestinal cells (FHs cells).

B. Removal of Surface Glycoproteins Alters (Decreases) Transport of MilkExosomes into Human and Other Mammalian Cells

(i) Removal of Surface Glycoproteins

Studies showed that the uptake of bovine milk exosomes into human andrat intestinal cells depended on surface proteins in both exosomes andcells. FIG. 38 shows a scheme for exosome processing to removeglycoproteins on the surface of milk exosomes. To remove surface proteinfrom milk exosomes, the exosomes were subjected to various proteases,including trypsin, proteinase K, AspN, GluC, ArgC. As shown in FIG. 39,trypsin cleaves at lysine or arginine residues; chymotrypsin cleaves ataromatic amino acids (cleaves on the C-terminal phenylalanine,tryptophan, and tyrosine) on peptide chains; lys-C cleaves at theC-terminal side of lysine residues; Arg-C cleaves at arginine and lysineresidues; Glu-C cleaves at glutamic acid and aspartic acid residues; andAsp-N cleaves at aspartic acid residues. Table A below shows thespecificities of various proteases. Following protease treatment, theexosomes were subject to ultracentrifugation. The supernatant containingthe enzyme cleaved surface peptides were identified using LC-MS/MS usingLC/MS-MS and Mascot, and glycoproteins were identified using SwissProt.One way Anova and Bonferroni's Multiple comparison was used to test forstatistical significance. The pellet containing the exosomes with itssurface proteins removed was resuspended in 1×PBS and used for thetransport studies shown in FIGS. 40-43.

TABLE A Proteases and Their Specificities Protease Terminal Name FamilyCleavage Site Cleavage pH Trypsin Serine Protease Lysine or Arginine C 8(except when either is followed by proline) Asp N MetalloproteaseAspartic Acid N 8 Glu C Serine Protease Glutamic acid and C 8 AsparticAcid Arg C Cysteine Protease Arginine when C 8 followed by proline andalso Lysine Chymotrypsin Serine Protease Aromatic amino acids C 8(phenylalanine, tryptophan, and tyrosine) Lys-C Serine Protease Lysineresidues C 8(ii) Transport Studies Show Decreased Transport Using Exosomes and Cellswith Surface Proteins Removed

The uptake of bovine milk exosomes into human and rat intestinal cellsdepended on surface proteins in both exosomes and cells. Exosomes weretreated with proteases as described above and depicted in FIG. 39. Whereapplicable, exosomes were treated with the proteases Glu-C (targetingglu), trypsin (targeting arg and lys), Glc C (targeting glutamic acidand aspartic acid), AspN (targeting aspartic acid), ArgG (targetingarginine and lysine), or proteinase K (non-specific); controls were nottreated with proteases. Exosome uptake was analyzed by measuring thecell fluorescence at 515 (excitation) and 640 nm (emission) using aBiotek FLx800 plate reader as previously described above.

When surface proteins were removed from exosomes or Cacos-2 cells viatreatment with proteinase K (100 mg/mL), exosome uptake decreased to32%±25% (exosome treatment) and 18%±16% (CaCo2 cell treatment) ofcontrols (P<0.05; n=3) (data not shown). FIG. 40B shows the effect onexosome uptake in CaCo2 cells after treatment of milk exosomes withtrypsin or Glu-C (FIG. 40B) or Arg-C or Asp-N FIG. 40C), demonstrating asignificant decrease in exosome transport into CaCo-2 cells when surfaceproteins were removed from the exosome. Studies using rat intestinalIEC-6 cells showed that milk exosomes treated with 0.105 mMol/L trypsinfor 30 mins demonstrated a decrease in transport into IEC-6 cells.Exosome uptake decreased to 82%±8% of controls (P,0.05; n=3). (Data notshown). FIG. 40A shows that surface proteins played an important role infacilitating exosome uptake into HUVECs. When exosomal surface proteinswere removed by treatment with proteinase K, exosome uptake decreased to˜50% of controls (FIG. 40A).

FIGS. 41A and 41B show exosome uptake by Caco-2 cells after treatment ofthe exosomes (FIG. 41A) or treatment of the CaCo-2 cells (FIG. 41B) withprotease(s) trypsin, Glc C, Arg C, Asp N, or a mixture thereof (n=3;p<0.05). As shown, treatment of exosomes with protease decreases theuptake of cow's milk exosomes in CaCo-2 cells by greater than 50%.Likewise, treatment of Caco-2 cells with protease decreases the uptakeof cow's milk exosomes in CaCo2 cells by greater than 50%. *P<0.05 vs.control. (N=3, means±S.D.).

FIGS. 42A and 42B show the effects on exosome transport in human smallintestinal FH cells after treatment with a protease (n=3; p<0.05).Protease treatment of exosomes (FIG. 42A) results in a decrease in theuptake of cow's milk exosomes in FH cells. Protease treatment of FHcells (FIG. 42B) with protease decreases the uptake of cow's milkexosomes in FH cells. *P<0.05 vs. control. (N=3, means±S.D.).

FIGS. 43A and 43B show the effects on exosome transport in humanmacrophage U937 cells after treatment with a protease (n=3; p<0.05).Protease treatment of exosomes (FIG. 43A) results in a decrease in theuptake of cow's milk exosomes in U937 cells. Protease treatment of U937cells (FIG. 43B) with protease decreases the uptake of cow's milkexosomes in U937 cells. *P<0.05 vs. control. (N=3, means±S.D.).

C. Altered Transport of Milk Exosomes into Human and Other MammalianCells by Removal of Glycans from Surface Glycoproteins(i) Removal of Glycans from Surface Glycoproteins

FIG. 48 shows a scheme for exosome processing to remove glycans fromglycoproteins on the surface of milk exosomes. To remove glycans fromthe surface protein of milk exosomes, the exosomes were subjected tovarious glycosidases, including neuraminidase, O-glycosidase,β-galactosidase, β-N-acetylglucosaminidase, and PNGaseF. As shown inTable B, neuraminidase cleaves all branched and unbranched chain sialicacids, O-glycosidase cleaves serine and threonine linked unsubstitutedGal-β (1→3)-GalNAc, β (1→4)-galactosidase cleaves β (1→4)-linkednonreducing terminal galactose, β-N-acetylglucosaminidase cleavesnon-reducing terminal β-linked Nacetylglucosamine residues, and PNGaseFwhich cleaves asparagine-linked complex, hybrid, or high mannoseoligosaccharides (except when fucosylated). Following glycosidasetreatment, the oligosaccharide fraction is removed for testing usingLC/MS-MS.

The exosomes with one or more types of glycans removed from its surfaceproteins were subject to ultracentrifugation. The pellet containing theexosomes with its one or more types of glycans removed was resuspendedin 1×PBS and used for the transport studies shown in FIGS. 46-48, 50,and 51.

TABLE B Temper- Time ature Enzyme Name Cleavage Site (hours) (° C.)A-(2→3,6,8,9)- All branched and 12 37 Neuraminidase unbranched sialicacids O-Glycosidase Serine or Threonine linked 12 37 unsubstitutedGal-β(1→3)- GalNAc N-Glycosidase N-linked glycans, including 12 37glycans with α(1,3)-linked core fucose β(1→4)-Galactosidaseβ(1→4)-linked nonreducing 12 37 terminal galactose β-N- Nonreducingterminal β- 12 37 Acetylglucosaminidase linked N-acetylglucosamine(exoglycosidase) PNGase F Asparagine linked complex, 12 37(glycoamidase) hybrid, or high mannose oligosaccharides (except whenfucosylated) bond between GlcNAc and Asn

(ii) Predicted Glycan Binding Sites on Identified Exosome MembraneProteins

FIG. 2 shows several membrane proteins identified in the membranefraction of milk exosomes, e.g., ALIX, CD9, and CD63. FIG. 44 shows atable of exemplary enzyme treatments, the expected number ofTransmembrane Helices (TMHs), and the predicted number of glycan bindingsites on identified exosome surface proteins in the presence of specificprotease treatment versus total deglycosylation (T.D). FIG. 45 shows aVenn diagram comparison for identified membrane proteins after specificprotease treatment versus specific protease treatment and total glycanremoved. LC/MS-MS studies identified 4 N-, 2 O-, and 2 C-glycosylatedproteins on the milk exosome surface. The presence of greater numbers ofTMHs after treatments that remove glycans suggests that the glycans wereindeed presented on the surface of the exosomes.

(iii) Transport Studies Show Decreased Transport Using Exosomes andCells with Glycans Removed from Surface Proteins

The uptake of bovine milk exosomes into human and rat intestinal cellsdepended on glycan content in membrane proteins found on the surface ofboth exosomes and cells. Exosomes were treated with glycosidases asdescribed above and depicted in Table 3. For example, exosomes and cellswere treated with the glycosidases A-(2→3,6,8,9)-Neuraminidase whichcleaves branched and unbranched sialic acids, O-Glycosidase whichcleaves serine or threonine linked unsubstituted Gal-β (1→3)-GaNAc,N-Glycosidase which cleaves N-linked glycans, β(1→4)-Galactosidase whichcleaves β(1→4)-linked nonreducing terminal galactose,β-N-Acetylglucosaminidase which cleaves nonreducing terminal β-linkedN-acetylglucosamine, and PNGase F which cleaves asparagine linkedcomplex, hybrid, or high mannose oligosaccharides (except whenfucosylated). Exosome uptake was analyzed by measuring the cellfluorescence at 515 (excitation) and 640 nm (emission) using a BiotekFLx800 plate reader (BioTek Instru-ments) as previously described above.The following studies demonstrated that exosome uptake is decreasedfollowing glycan removal from surface proteins of exosomes or mammaliancells (via treatment with a glycosidase). FIG. 46 shows exosome uptakein Caco2 cells following enzymatic removal of glycan from exosomesurface proteins using PNGase, β-galactosidase, O-glycosidase,N-acetyl-glucosamidase, or a mixture thereof. Removal of glycan resultsin a decrease in exosome uptake in Caco-2 cells. FIGS. 47 A and 47B showexosome uptake in Caco2 cells following enzymatic removal of glycan fromsurface proteins in exosomes and CaCo-2 cells, respectively usingβ-N-acetyl-glucosamidase, PNGase F, β-galactosidase, O-glycosidase,neuraminadase, or a mixture thereof. Removal of glycan from eitherexosomes or CaCo-2 cells results in a decrease in exosome uptake inCaco-2 cells. (both studies *P<0.05 vs. control; N=3, means±S.D.) FIGS.48 A and 48B show exosome uptake in human small intestinal FH cellsfollowing enzymatic removal of glycan from surface proteins in exosomesand FH cells, respectively using β-N-acetyl-glucosamidase, PNGase F,β-galactosidase, O-glycosidase, neuraminadase, or a mixture thereof.Removal of glycan from either exosomes or FH cells results in a decreasein exosome uptake in FH cells. (both studies *P<0.05 vs. control; N=3,means±S.D.) FIGS. 49 A and 49B show exosome uptake in U937cellsfollowing enzymatic removal of glycan from surface proteins in exosomesand U937 cells, respectively using β-N-acetyl-glucosamidase, PNGase F,β-galactosidase, O-glycosidase, neuraminadase, or a mixture thereof.Removal of glycan from exosomes results in a decrease in exosome uptakein U937 cells. Removal of glycan from U937 cells results in a decreasein exosome uptake in U937 cells following treatment withβ-N-acetyl-glucosamidase, neuraminidase, and a mixture of glycosidases.(both studies *P<0.05 vs. control; N=3, means±S.D.)

(iv) Use of Lectins to Identify Glycans on the Surface of Milk Exosomes

FIGS. 50A-D show Eastern/Lectin Blots that identify glycans present onthe membranes of milk exosomes. NE—Normal Exosome; CE—CytoplasmicExtract; MP—Membrane Protein. FIG. 50A shows a lectin blot using Con A,which is specific for alpha linked mannose, as a probe. FIG. 50B shows alectin blot using PNA, which is specific for Gal β 1-3 GalNAc 1 Ser/Thr,as a probe. FIG. 50C shows a lectin blot using SBA, which is specificfor GalNAc, as a probe. FIG. 50D shows a lectin blot using SNA, which isspecific for sialic acid, as a probe. These results demonstrate thepresence of alpha linked mannose, Gal β 1-3 GalNAc 1 Ser/Thr, GalNAc,and sialic acid glycans in the membranes of exosomes.

TABLE C Lectin Symbol Lectin Source Ligand Motif Mannose-Binding LectinsConA Concanavalin A Canavalia α-D-mannosyl and ensiformis α-D-glucosylresidues branched α-mannosidic structures (high α- mannose type, orhybrid type and biantennary complex type N-Glycans) LCH Lentil lectinLens Fucosylated core culinaris region of bi- and triantennary complextype N-Glycans GNA Snowdrop lectin Galanthus α 1-3 and α 1-6 linkednivalis high mannose structures Galactose/N-acetylgalactosamine bindinglectins RCA Ricin, Ricinus Ricinus Galβ1-4GalNAcβ1-R communis communisAgglutinin, RCA120 PNA Peanut Arachis Galβ1-3GalNAcα1-Ser/ agglutininhypogaea Thr (T-Antigen) AIL Jacalin Artocarpus (Sia)Galβ1-3GalNAcα1-integrifolia Ser/Thr (T-Antigen) VVL Hairy vetch Vicia villosaGalNAcα-Ser/Thr lectin (Tn-Antigen) DBA Dolichos α-linkedN-acetylgalactosamine biflorus agglutinin SBA Soybean soybean terminal -and agglutinin -N-acetylgalactosamine and galactopyranosyl residuesN-acetylglucosamine binding lectins WGA Wheat Germ TriticumGlcNAcβ1-4GlcNAcβ1- Agglutinin, vulgaris 4GlcNAc, Neu5Ac WGA (sialicacid) PHA-E Phaseolus Red kidney terminal galactose, vulgaris beanN-acetylglucosamine and agglutinin mannose residues of complex glycansN-acetylneuraminic acid binding lectins SNA Elderberry SambucusNeu5Acα2-6Gal(NAc)-R lectin nigra MAL Maackia MaackiaNeu5Ac/Gcα2,3Galβ1, amurensis amurensis 4Glc(NAc) leukoagglutinin MAHMaackia Maackia Neu5Ac/Gcα2,3Galβ1, amurensis amurensis3(Neu5Acα2,6)GalNac hemoagglutinin Fucose binding lectins UEA Ulexeuropaeus Ulex Fucα1-2Gal-R agglutinin europaeus AAL Aleuria aurantiaAleuria Fucα1-2Galβ1-4(Fucα1- lectin aurantia 3/4)Galβ1-4GlcNAc,R2-GlcNAcβ1-4(Fucα1- 6)GlcNAc-R1

(v) Lectin Blockage of Glycans on the Surface Membrane of ExosomesResults in Decreased Transport in FH Cells

The uptake of bovine milk exosomes into human small intestinal FH cellsdepended on glycan function in membrane proteins found on the surface ofboth exosomes and cells. In this transporter assay study, variouslectins were used to bind to and block certain glycans. Exosome uptakewas analyzed by measuring the cell fluorescence at 515 (excitation) and640 nm (emission) using a Biotek FLx800 plate reader (BioTekInstruments) as previously described above. FIGS. 51A and 51B show theresults of a lectin blocking study in which exosomes or FH cells weretreated with various lectins, including ConA (binds to α-D-mannosyl andα-D-glucosyl residues), PHA-E (binds to terminal galactose,N-acetylglucosamine and mannose residues of complex glycans), SNA (bindsto Neu5Acα2-6Gal(NAc)-R), PNA (binds to Galβ1-3GalNAcα1-Ser/Thr), WGA(binds to GlcNAcβ1-4GcNAcβ1-4GcNAc, Neu5Ac), RCA (binds toGalβ1-4GalNAcβ1-R), DBA (binds to α-linked N-acetylgalactosamine), SBA(binds to terminal- and -N-acetylgalactosamine and galactopyranosylresidues), MAL (binds to Neu5Ac/Gcα2,3Galβ1,4Glc(NAc)) and UEA (binds toFucα1-2Gal-R). The blocking of glycans present on exosomes or on FHcells (FIG. 51A and FIG. 51B, respectively) with lectin decreases theuptake of cow's milk exosomes in FH cells. (*P<0.05 vs. control; N=3,means±S.D.).

D. Inhibition of Endocytosis, Vesicle Trafficking, and CarbohydrateBlocking Results in Decreased Exosome Transport into Cells

Exosome transport was measured in HUVEC cells (expressed as ng exosomalprotein/81,750 cells/h) pretreated for 30 min with 10 mg/mL Cyt D, 20mg/mL BFA or 150 mmol/L carbohydrate competitors (glucose, galactose),using an exosome concentration of 55 mg/200 mL (n=5). Treatment with CytD, BFA, and carbohydrate competitors (glucose, galactose) was continuedfor the duration of the transport studies. Exosome uptake was analyzedby measuring the cell fluorescence at 515 (excitation) and 640 nm(emission) using a Biotek FLx800 plate reader (BioTek Instruments) aspreviously described above. FIG. 52 shows the results of an exosometransport study in which inhibitors of endocytosis (cytochalasin D=CytD), vesicle trafficking (brefeldin A=BFA), and carbohydrate blockage(glucose, galactose) were shown to cause a decrease in exosome uptake inHUVEC cells. *Different from control, P, 0.05. Values are means 6 SDs.Specifically, exosome transport into HUVEC cells decreased to <50% ofcontrols (FIG. 52), indicating that exosomes are trafficked to HUVEC andtransported into cells via endocytosis. Also, the carbohydratecompetitor galactose, but not glucose, caused a significant decrease inexosome uptake indicating that galactose can compete with surfaceglycans on milk exosomes that mediate transport.

Results

417 exosomal surface proteins were identified, including N-glycans (4),O-glycans (2), and C-glycans (2). When exosomes were treated with Glu-Cor trypsin, transport rates (Vmax) decreased compared with controls(arbitrary units): 88±1 for control, 40±0.6 for trypsin, 19±0.3 forGlu-C (P<0.05 vs. control). For comparison, 504 proteins were identifiedin breast milk exosomes (6-N-glycans, 4 O-glycans, and 3 C-glycans). Theidentities of surface glycoproteins were distinct in bovine and humanmilk exosomes.

CONCLUSION

Eight glycoproteins identified on the surface of cow's milk exosomesappear to be essential for intestinal transport. Glycan features, asopposed to protein features, are important for exosome recognition byintestinal cells.

Example 9: Animal Studies Showing that Modification of Glycans Found onMilk Exosomes Alter the Uptake of Exosomes into Mammalian Tissues A. InVivo Transport of Exosomes Having Altered Glycan in Surface Proteins

Milk exosomes having altered glycan in their surface proteinsdemonstrate altered uptake in various tissues in vivo. Milk exosomeswere isolated as described in Example 1. A. The milk exosomes weretreated with a glycosidase, for example, A-(2→3,6,8,9)-Neuraminidase,O-Glycosidase, N, -Glycosidase, β(1→4)-Galactosidase,β-N-Acetylglucosaminidase, PNGase F, as described above in Example 1. E(i) and as shown in FIG. 55A. For example, in some embodiments, allbranched and unbranched sialic acids are removed. In some embodiments,serine or threonine linked unsubstituted Gal-β (1→3)-GalNAc glycans areremoved. In some embodiments, N-linked glycans are removed. In someembodiments, glycans with α(1,3)-linked core fucose are removed. In someembodiments, glycans with β(1→4)-linked nonreducing terminal galactoseare removed. In some embodiments, glycans with nonreducing terminalβ-linked N-acetylglucosamine are removed. In some embodiments,asparagine linked complex, hybrid, or high mannose oligosaccharides areremoved.

In one specific example, PnGase F was used to remove asparagine-linkedcomplex, hybrid, or high mannose oligosaccharides from the surfaceproteins of milk exosomes. FIG. 55A shows a scheme for exosomeprocessing to remove certain glycans from membrane proteins on thesurface of exosomes, e.g., asparagine-linked complex, hybrid, or highmannose oligosaccharides (via the use of PnGase F). Milk exosomes withtheir native surface proteins (control) and milk exosomes having alteredglycan in their surface proteins were separately ultracentrifuged topellet the exosomes and the pelleted exosomes were then labeled withDiR. 1,1′dioctadecyl-3, 3, 3′, 3′-tetramethylindotricarbocyanineiodide,DiR, is a lipophilic tracer that can be used to label lipoproteins inliving or fixed tissues which does not affect cell viability,development, or basic physiological properties (Invitrogen). TheDiR-labelled control exosomes and DiR-labelled exosomes with alteredglycan were separately administered via oral gavage to wild-type mice(n=3 for each exosome group). After 12 hours, the mice were sacrificedand various tissues were examined using fluorescence. FIG. 25B shows theresults of a fluorescence study in which fluorescent-labelled exosomeshaving native glycosylation (control) versus altered glycosylation(removal of asparagine-linked complex, hybrid, or high mannoseoligosaccharides via the use of PnGase F) were compared. As shown inFIG. 55B, control exosomes exhibited significant uptake in varioustissues as determined by the bright fluorescence observed in heart,lung, spleen, and liver with little accumulation in stomach and smallintestine. In contrast, the glycan-altered exosomes showed significantlydecreased uptake in heart, lung, spleen, and liver and significantlyincreased accumulation of non-absorbed exosomes in the small intestine,as determined by fluorescence intensity. This indicates that thepresence of asparagine-linked complex, hybrid, or high mannoseoligosaccharides on milk exosomes facilitates the uptake of milkexosomes from the gastrointestinal tract for delivery to heart, lung,spleen and liver and. In contrast, the removal of asparagine-linkedcomplex, hybrid, or high mannose oligosaccharides on milk exosomesresults in a loss of transport from the gastrointestinal tract totissues compared with milk exosomes having native glycosylation.

B. Production of Transgenic Knock-Out Mice Having Deletion of DifferentGlycan Transferase Genes

Bmi1^(tm1(cre/ERT)Mrc)/J mice to generate transgenic conditional(tamoxifen-inducible) knockout mice homozygous for deletion of a glycantransferase gene (FIG. 53A). FIG. 54B shows the Cre-LoxP mediated genedeletion method used to knock-out the glycan transferase gene. Cre-LoxPrecombination is a well-known technique in which recombination betweenLoxP sites is catalysed by Cre recombinase. Floxing a gene of interest(sandwiching the gene between two LoxP sites) allows the gene to bedeleted (knocked out), translocated or inverted via Cre-Loxrecombination. Nagy A at al., (2000). “Cre recombinase: the universalreagent for genome tailoring.” Genesis. 26 (2): 99-109. FIG. 54B depictsthe Cre-LoxP mediated gene deletion method used to knock-out the glycantransferase gene.

Floxed genes can also be used to produce tissue-specific knockout miceby using the Cre recombinase with a tissue-specific promoter whichcauses the floxed gene to be inactivated (knocked out) only in thespecific targeted tissue. Cre-Lox knockouts can also be inducible, forexample, using tamoxifen to induce Cre recombinase. In this case, Crerecombinase is fused to a portion of the mouse estrogen receptor, whichis naturally localized to the cytoplasm via its interactions withchaperone proteins such as heat shock protein 70 and 90. Tamoxifen bindsto the estrogen receptor and disrupts its interactions with thechaperones which allows the Cre-estrogen receptor fusion protein toenter the nucleus and perform recombination on the floxed gene. Hayashi,Shigemi; McMahon, Andrew P. (2002). “Efficient Recombination in DiverseTissues by a Tamoxifen-Inducible Form of Cre: A Tool for TemporallyRegulated Gene Activation/Inactivation in the Mouse”. DevelopmentalBiology. 244 (2): 305-318. Using this process, severaltamoxifen-inducible conditional glycan transferase knockout mice weremade (FIGS. 53 and 54). Tamoxifen-inducible conditional glycantransferase knockout mice having a glycan transferase gene deleted orotherwise knocked out (see Table D below) are made. For example,transgenic knockout mice having a conditionally deleted glucosaminyl(N-acetyl) transferase 3, mucin type (GCNT3); O-linkedβ-N-acetylglucosamine transferase (OGT); Protein-O-fucosyl transferase(PoFUT1); or Mannoside acetyl glucosaminyltransferase (MGAT1) genedeleted were made. FIG. 54B shows the genotyping results for transgenicmice having a deleted MGAT1 or PoFUT1 gene, showing the knockout ofthese respective genes. The transgenic mice are used to study or monitorthe effect of specific glycosylation on exosome transport to varioustissues.

TABLE D Knockout Strain Glycan/Enzyme/Loss B6.129- JL Glucosaminyl(N-Acetyl) Gcnt3^(tmlJxm)/J Transferase 3, mucin type (beta-6-N-acetylglucosamine-transferase) catalyzes the formation of core 2 andcore 4 O- glycans on mucin-type glycoproteins loss of core 2 and 4O-glycans OGT ™ JH UDP-N-acetylglucosamine—peptide N-acetylglucosaminyltransferase (EC 2.4.1.255) (O-linkedβ-N-acetylglucosamine transferase; O-GlcNAc-transferase) catalyzes theaddition of a single N- acetylglucosamine in O-glycosidic linkage toserine or threonine residues loss of O-glycans; loss of O-GlcNAcylationPofut1 ™ PS Protein-O-fucosyl transferase adds O-fucose through anO-glycosidic linkage to conserved serine or threonine residues loss ofO-fucose glycans B6.129S2- PS Alpha-1,3-mannosyl-glycoprotein 2-beta-N-Mgat1 ™ acetylglucosaminyltransferase (Mannoside acetylglucosaminyltransferase) synthesis of hybrid and complex N-glycans lossof N-glycans

C. Exosome Transport Using Transgenic Knock-Out Mice Having Deletion ofDifferent Glycan Transferase Genes

Tamoxifen-inducible conditional glycan transferase knockout mice(homozygous) are made as described above. FIGS. 54A and 54B show variousgene knockouts which result in alteration of glycosylation andcorresponding genotyping results. Differences in milk exosome uptake inthese knockout mice as compared with wild type mice can be used tofurther identify or characterize the glycosylation of surface proteinsinvolved in exosome uptake, as well as the glycan transferase enzymesinvolved in exosome uptake. Such characterization is useful for thedesign of modified exosomes with improved stability, improved uptake,altered uptake, targeted uptake and/or improved delivery of cargo.

Exosomes are isolated as described in Example 1. A.Fluorescence-labelled (DiR) is added to the exosome pellet. TheDiR-labelled exosomes are administered via oral gavage to wild-type mice(n=3) and tamoxifen-inducible conditional glycan transferase knockoutmice (n=3) having a conditional deletion in a glycan transferase gene(or other gene involved in glycosylation), for example, GCNT3 gene, OGTgene, PoFUT1 gene, or MGAT1 gene. After 12 hours, the wild-type mice andtransgenic mice are sacrificed and various organs (e.g., heart, lungs,liver, spleen, kidney, intestine, stomach, etc) are examined viafluorescence. Fluorescent-labelled exosomes having native glycosylation(control wild-type mice) versus fluorescent-labelled exosomes havingaltered glycosylation (e.g., lacking core 2 and 4 O-glycans, lackingO-GcNAcylation or O-glycans, lacking O-fucose glycans, or lackingN-glycans) as found in the transgenic mice are compared to determine theeffect of altered glycosylation on the uptake of exosomes, e.g., milkexosomes, in vivo. These methods provide other means for determining ormonitoring exosome uptake of milk exosomes having altered glycan in itssurface proteins. Such characterization is useful for the design ofmodified exosomes with improved stability, improved uptake, altereduptake (increased or decreased), targeted uptake and/or improveddelivery of cargo. The transgenic mice can also be used as a means ofproducing milk exosomes having various altered glycosylation of itssurface proteins.

Example 10: Method of Loading Exosomes with an Exogenous Cargo

Microvesicles of the present disclosure, including milk exosomes, can beloaded with cargos using various methods known in the art such aselectroporation or transfection with cationic lipid reagents. Othermethods include loading by ultracentrifugation. For example, proceduresfor loading cargos are provided in U.S. Pat. No. 9,085,778, US2016/0000710, and WO 2015/161184, each of which is hereby incorporatedby reference. Additional literature on loading methods for exosomesincludes Luan, X. et al., Acta Pharmacol Sin. 2017 June; 38(6): 754-763and Munagala R, et al., Cancer Lett. 2016 Feb. 1; 371(1):48-61, each ofwhich is hereby incorporated by reference.

Those procedures include i) suspending therapeutic agents in PEG-400,mixing with milk-derived exosomes, followed by low-speed centrifugation;ii) dissolving therapeutic agents in ethanol, mixing with milk- orcolostrum-derived exosomes, low-speed centrifugation (10,000×g) toremove unbound therapeutic agent, and finally high-speed centrifugation;and iii) mixing therapeutic agents in ethanol with 100,000 whey(obtained after the 100,000×g centrifugation), low-speed centrifugationand finally 120,000×g centrifugation. In one procedure, incubation ofexosomes in PBS with test agents in the presence of 10% ethanol or 10%ethanol:acetonitrile (1:1) will load therapeutic agents into theexosomes. Sucrose density gradient ultracentrifugation can be used toconfirm the presence of drugs embedded in the exosomes. Testing for invitro release may be performed using dialysis tubes against buffercontaining the surfactant Tween-80 at 37° C.

OTHER EMBODIMENTS

It is to be understood that while the disclosure has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of thedisclosure, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A milk exosome comprising: a biological membrane surrounding a lumen,wherein the biological membrane comprises one or more glycoprotein(s),wherein the biological membrane is modified as compared with the naturalbiological membrane of the milk exosome.
 2. The milk exosome of claim 1,wherein the biological membrane is modified such that it has anincreased number of one or more of its native glycoprotein(s).
 3. Themilk exosome of claim 1, wherein the biological membrane is modifiedsuch that it has a decreased number of one or more of its nativeglycoprotein(s).
 4. (canceled)
 5. (canceled)
 6. The milk exosome ofclaim 1, wherein the biological membrane is modified such that one ormore of its native glycoprotein(s) is not present.
 7. The milk exosomeof claim 1, wherein the biological membrane is modified such that itincludes one or more glycoprotein(s) that is not naturally present inthe natural biological membrane.
 8. The milk exosome of claim 1, whereinthe biological membrane is modified such that one or more of its nativeglycoprotein(s) is altered.
 9. The milk exosome of claim 8, wherein theone or more native glycoprotein(s) is altered such that the number ofglycan residues present on the glycoprotein(s) is increased.
 10. Themilk exosome of claim 9, wherein the exosome is produced usingglycosylation that adds one or more glycans to the glycoprotein.
 11. Themilk exosome of claim 8, wherein the one or more native glycoprotein(s)is altered such that the number of glycan residues present on theglycoprotein(s) is decreased. 12-15. (canceled)
 16. The milk exosome ofclaim 8, wherein the one or more native glycoprotein(s) is altered suchthat it comprises a modified glycan.
 17. The milk exosomes of claim 16,wherein the modified glycan comprises at least one carbohydrate moietythat differs from that of the glycan in the native glycoprotein(s). 18.The exosome of claim 16, wherein the modified glycan comprises one ormore D- or L-glucose, erythrose, fucose, galactose, mannose, lyxose,gulose, xylose, arabinose, ribose, 2′-deoxyribose, glucosamine,lactosamine, polylactosamine, glucuronic acid, sialic acid, sialyl-LewisX (SLex), N-acetyl-glucosamine, N-acetyl-galactosamine, neuraminic acid,N-glycolylneuraminic acid (Neu5Gc), N-acetylneuraminic acid (Neu5Ac), anN-glycan chain, an O-glycan chain, a Core 1, Core 2, Core 3, or Core 4structure, or a phosphate- or acetate-modified analog thereof or acombination thereof. 19-25. (canceled)
 26. The exosome of claim 1,wherein uptake of the milk exosome into a mammalian cell is altered ascompared with the uptake of a corresponding milk exosome having itsnatural biological membrane.
 27. The exosome of claim 26, wherein uptakeof the milk exosome into a mammalian cell is increased.
 28. The exosomeof claim 26, wherein the mammalian cell is selected from an intestinalcell, venous endothelial cell or other endothelial cell, immune cell,macrophage, intestinal mucosa, peripheral cell of the liver, spleen,lung, brain, kidneys, or pancreas, cancer cell, or fetal cell. 29.(canceled)
 30. The exosome of claim 1, wherein the milk exosome istargeted to a specific mammalian cell or tissue.
 31. The exosome ofclaim 30, wherein the mammalian cell is selected from an intestinalcell, venous endothelial cell or other endothelial cell, immune cell,macrophage, intestinal mucosa, peripheral cell of the liver, spleen,lung, brain, kidneys, or pancreas, cancer cell, or fetal cell.
 32. Theexosome of claim 30, wherein the mammalian tissue is selected fromliver, spleen, lung, brain, kidneys, pancreas, gastrointestinal tract,small intestine, colon, stomach, or heart.
 33. (canceled)
 34. Theexosome of claim 1, wherein the stability of the milk exosome in thegastrointestinal tract, systemic circulation, lymphatic circulation,intracellular conditions, or other tissues or organs of a subject isincreased as compared with an exosome having its natural biologicalmembrane.
 35. The exosome of claim 1, wherein the stability of the milkexosome under physiological conditions in a subject is increased ascompared with a milk exosome having its natural biological membrane. 36.The milk exosome of claim 1, further comprising an exogenous cargoencapsulated in said lumen.
 37. The milk exosome of claim 1, wherein themilk exosome further comprises an miRNA or mRNA that is biologicallyactive in a mammal.
 38. The milk exosome of claim 1, wherein the milkexosome is isolated from sheep, goat, camel, horse, donkey, reindeer,yak, buffalo, or bovine (cow) milk or colostrum.
 39. The milk exosome ofclaim 36, wherein the exogenous cargo is selected from one or morenucleic acid molecules, polypeptides, lipids, vitamins, minerals, smallmolecules, pharmaceuticals, hormones, or enzymes.
 40. The milk exosomeof claim 36, wherein the exogenous cargo comprises a therapeutic agent.41. The milk exosome of claim 40, wherein the therapeutic agent isselected from mRNAs, polypeptides, miRNAs, miRNA antagonists, nutrients,antibiotics, cancer drugs, activators of Toll-like receptors, ormolecules capable of delivery to macrophages.
 42. The milk exosome ofclaim 40, wherein the therapeutic agent is a cancer drug selected from achemotherapeutic, an immunotherapeutic, a hormone therapeutic, or atargeted therapeutic. 43-52. (canceled)
 53. A method of altering theuptake of a milk exosome into a mammalian cell or tissue, the milkexosome having a biological membrane comprising one or moreglycoprotein(s), comprising modifying the biological membrane of theexosome.
 54. The method of claim 53, wherein the uptake of the milkexosome into a mammalian cell or tissue is increased.
 55. The method ofclaim 53, wherein the uptake of the milk exosome into a mammalian cellor tissue is decreased.
 56. The method of claim 53, wherein the uptakeof the milk exosome into a mammalian cell or tissue is selectivelyincreased in a targeted mammalian cell or tissue.
 57. The method ofclaim 53, wherein the uptake of the milk exosome into a mammalian cellor tissue is selectively decreased in a targeted mammalian cell ortissue.
 58. A method of targeting a milk exosome to a selected mammaliancell or tissue, the milk exosome having a biological membrane comprisingone or more glycoprotein(s), comprising modifying the biologicalmembrane of the milk exosome.
 59. The method of claim 53, wherein thebiological membrane is modified such that it has an increased number ofone or more of its native glycoprotein(s).
 60. The method of claim 53,wherein the biological membrane is modified such that it has a decreasednumber of one or more of its native glycoprotein(s). 61-63. (canceled)64. The method of claim 53, wherein the biological membrane is modifiedsuch that it includes one or more glycoprotein(s) that is not naturallypresent in the natural biological membrane.
 65. The method of claim 53,wherein the biological membrane is modified such that one or more of itsnative glycoprotein(s) is altered.
 66. The method of claim 65, whereinthe one or more native glycoprotein(s) is altered such that the numberof glycan residues present on the glycoprotein(s) is increased.
 67. Themethod of claim 65, wherein the one or more native glycoprotein(s) isaltered such that the number of glycan residues present on theglycoprotein(s) is decreased. 68-76. (canceled)
 77. The method of claim53, wherein the mammalian cell is selected from an intestinal cell,venous endothelial cell or other endothelial cell, immune cell,macrophage, intestinal mucosa, peripheral cell of the liver, spleen,lung, brain, kidneys, or pancreas, cancer cell, or fetal cell. 78-97.(canceled)